Chemical compounds

ABSTRACT

Disclosed are compounds of Formula III. Also disclosed are salts of the compounds, pharmaceutical composition comprising the compounds or salts, and methods for treating HCV infection by administration of the compounds or salts.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed as a continuation application of U.S. Ser. No.13/677,358, filed on Nov. 15, 2012, which is a continuation applicationof U.S. Ser. No. 12/936,545, filed Oct. 6, 2010 (now U.S. Pat. No.8,344,155), which is a National Phase Application of InternationalApplication No. PCT/US2010/046782 filed on Aug. 26, 2010, which claimspriority from 61/239,855 filed on Sep. 4, 2009, 61/297,324 filed on Jan.22, 2010 and 61/348,767 filed on May 27, 2010 in the United States, allof which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present disclosure relates to antiviral compounds. In particular,the present disclosure relates to compounds useful for the treatment ofhepatitis C virus (HCV) infection, crystalline salts of the compounds,pharmaceutical compositions comprising the compounds, and methods fortreating HCV infection.

BACKGROUND OF THE INVENTION

Chronic infection with HCV is a major health problem associated withliver cirrhosis, hepatocellular carcinoma and liver failure. Anestimated 170 million chronic carriers worldwide are at risk ofdeveloping liver disease. See, for example, Szabo, et al., Pathol.Oncol. Res. 2003, 9:215-221, and Hoofnagle J H, Hepatology 1997,26:15S-20S. In the United States alone 2.7 million are chronicallyinfected with HCV, and the number of HCV-related deaths in 2000 wasestimated between 8,000 and 10,000, a number that is expected toincrease significantly over the next years. Infection by HCV isinsidious in a high proportion of chronically infected (and infectious)carriers who may not experience clinical symptoms for many years. Livercirrhosis can ultimately lead to liver failure. Liver failure resultingfrom chronic HCV infection is now recognized as a leading cause of livertransplantation.

HCV is a member of the Flaviviridae family of RNA viruses that affectanimals and humans. The genome is a single 9.6-kilobase strand of RNA,and consists of one open reading frame that encodes for a polyprotein of3000 amino acids flanked by untranslated regions at both 5′ and 3′ ends(5′- and 3′-UTR). The polyprotein serves as the precursor to at least 10separate viral proteins critical for replication and assembly of progenyviral particles. The organization of structural and non-structuralproteins in the HCV polyprotein is as follows:C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b. Because the replicative cycle ofHCV does not involve any DNA intermediate and the virus is notintegrated into the host genome, HCV infection can theoretically becured. While the pathology of HCV infection affects mainly the liver,the virus is found in other cell types in the body including peripheralblood lymphocytes. See, for example, Thomson B J and Finch R G, ClinMicrobial Infect. 2005, 11:86-94, and Moriishi K and Matsuura Y,Antivir. Chem. Chemother. 2003, 14:285-297.

At present, the standard treatment for chronic HCV is interferon alpha(IFN-alpha) in combination with ribavirin and this requires at least six(6) months of treatment. IFN-alpha belongs to a family of naturallyoccurring small proteins with characteristic biological effects such asantiviral, immunoregulatory and antitumoral activities that are producedand secreted by most animal nucleated cells in response to severaldiseases, in particular viral infections. IFN-alpha is an importantregulator of growth and differentiation affecting cellular communicationand immunological control. Treatment of HCV with interferon hasfrequently been associated with adverse side effects such as fatigue,fever, chills, headache, myalgias, arthralgias, mild alopecia,psychiatric effects and associated disorders, autoimmune phenomena andassociated disorders and thyroid dysfunction. Ribavirin, an inhibitor ofinosine 5′-monophosphate dehydrogenase (IMPDH), enhances the efficacy ofIFN-alpha in the treatment of HCV. Despite the introduction ofribavirin, more than 50% of the patients do not eliminate the virus withthe current standard therapy of interferon-alpha (IFN) and ribavirin. Bynow, standard therapy of chronic hepatitis C has been changed to thecombination of pegylated IFN-alpha plus ribavirin. However, a number ofpatients still have significant side effects, primarily related toribavirin. Ribavirin causes significant hemolysis in 10-20% of patientstreated at currently recommended doses, and the drug is both teratogenicand embryotoxic. Even with recent improvements, a substantial fractionof patients do not respond with a sustained reduction in viral load andthere is a clear need for more effective antiviral therapy of HCVinfection. See, for example, Fried, et al. N. Engl. J Med 2002,347:975-982.

A number of approaches are being pursued to combat the virus. Theyinclude, for example, application of antisense oligonucleotides orribozymes for inhibiting HCV replication. Furthermore, low-molecularweight compounds that directly inhibit HCV proteins and interfere withviral replication are considered as attractive strategies to control HCVinfection. Among the viral targets, the NS3/4A protease/helicase and theNS5b RNA-dependent RNA polymerase are considered the most promisingviral targets for new drugs. See, for example, Ni, Z. J. and Wagman, A.S. Curr. Opin. Drug Discov. Devel. 2004, 7, 446-459, Beaulieu, P. L. andTsantrizos, Y. S. Curr. Opin. Investig. Drugs 2004, 5, 838-850, andGriffith, et al., Ann. Rep. Med. Chem 39, 223-237, 2004.

Besides targeting viral genes and their transcription and translationproducts, antiviral activity can also be achieved by targeting host cellproteins that are necessary for viral replication. For example, Watashi,et al, Molecular Cell, 19, 111-122, 2005, show how antiviral activitycan be achieved by inhibiting host cell cyclophilins. Alternatively, apotent TLR7 agonist has been shown to reduce HCV plasma levels inhumans. See, Horsmans, et al, Hepatology, 42, 724-731, 2005.

Compounds said to be useful for treating HCV infection are disclosed,for example, in WO 2008/064218 (Leivers et. al), WO 2008/244380 (Bachandet. al), and US 2009/0068140 (Bachand et. al). These references alsodisclose methods for preparing the compounds, compositions comprisingthe compounds, pharmaceutical compositions comprising the compounds andadditional compounds, methods of treating HCV, salts of the compounds,routes of administration, and other information regarding how to make,formulate, and use the compounds.

SUMMARY OF THE INVENTION

Briefly, in one aspect, the present invention discloses compounds ofFormula I;

wherein each R¹ is independently H or C₁₋₃alkyl;each R² is independently C₁₋₃alkyl;each X is independently CRR, O, or S;n is 2 or 3; andeach R is independently methyl, hydrogen, or deuterium.

In another aspect, the present invention discloses compounds of FormulaII;

wherein each R¹ is independently H or C₁₋₃alkyl;each R² is independently C₁₋₃alkyl;each X is independently CRR, O, or S;each n is independently 2 or 3; andeach R is independently methyl, hydrogen, or deuterium.

In another aspect, the present invention discloses compounds of FormulaIII;

wherein each R¹ is independently H or C₁₋₃alkyl;each R² is independently C₁₋₃alkyl;on each carbon to which there are R³ groups, either both R³s are H orthe R³ groups together with the carbon to which they are bonded form a4-, 5-, or 6-membered saturated spiro ring with the proviso that thereis no more than 1 spiro ring on each saturated nitrogen-containing ring;each saturated spiro formed from R³ groups is independently cycloalkyl,or may contain 1 or 2 oxygen atoms, or 1 or 2 sulfur atoms, or 1 SO₂, or1 NR⁴;each R⁴ is independently H, C(O)OC₁₋₄alkyl, C(O)C₁₋₄alkyl,C(O)NC₁₋₄alkyl, or SO₂C₁₋₄alkyl;each spiro ring may optionally be substituted with deuterium, fluorine,or 1 or 2 methyl groups.

In another aspect, the present invention discloses pharmaceuticallyacceptable salts of the compounds of Formula I, II, or III.

In another aspect, the present invention discloses pharmaceuticalcompositions comprising a compound of Formula I, II, or III, or apharmaceutically acceptable salt thereof.

In another aspect, the present invention discloses a method for treatinga viral infection, for example infection with HCV, in a human,comprising administration of a pharmaceutical composition of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

In the above Formulas I, II, and III, except for when R¹ is hydrogen,the carbon to which R¹ is attached is chiral. In addition, the depictedtertiary carbon in each of the two depicted nitrogen containing5-membered saturated heterocyclic rings is also chiral. Therefore, thecompounds contain at least two chiral carbon atoms and when each R¹ isC₁₋₃ alkyl, the compounds contain at least 4 chiral carbon atoms.Therefore, the compounds can exist in various enantiomeric mixtures.

In an embodiment of the invention, the compounds of Formula I, II, orIII, or pharmaceutically acceptable salts thereof, are enantiomericallyenriched with the enantiomer wherein all of the chiral carbons referredto in the previous paragraph are in the S configuration. In general,reference to an enantiomerically enriched compound or salt, is meant toindicate that the specified enantiomer will comprise more than 50% byweight of the total weight of all enantiomers of the compound or salt.An example of a compound with four chiral carbons in S configuration isillustrated below.

In an embodiment of the invention, each X is identical.

In an embodiment of the invention, either all Rs are H or all Rs aredeuterium (D). In other words, in an embodiment of the invention, eitherevery CRR group in the spiro is CH₂ or every CRR group in the spiro isCD₂. Deuterium is naturally present in very small amounts in hydrogencompounds. By designating a substituent as deuterium or D, applicantsmean that the natural isotopic amount of deuterium has been increased sothat more that half of that particular substituent is D as compared toH.

In an embodiment of the invention, no more than 2 Rs are methyl.

In an embodiment of the invention, in compounds of Formula III, when R³groups form a spiro ring on each saturated nitrogen-containing ring,each of said spiro groups is bonded to the same relative carbon atom ineach saturated nitrogen containing ring.

Pharmaceutically acceptable salts can be prepared by methods well knownin the art. Suitable salts include those described, for example, in P.Heinrich Stahl, Camille G. Wermuth (eds.), handbook of PharmaceuticalSalts properties, selection, and Use; 2002. See also, WO 2009/020828(Kimet. Al), which describes the preparation of crystalline salts ofcertain anti-viral compounds. Preferred salts include HCl salts, forexample a di-HCl salt, and sulphate salts.

The compounds and salts of the invention may be used alone or incombination with one or more other therapeutic agents. In one aspect thefurther therapeutic agent is selected from Standard of Care therapiessuch as interferon/ribavarin, small molecule HCV replication inhibitors(more commonly referred to as direct acting antivirals. Suitablecombination therapies are described, for example in WO 2008/064218(Leivers et. al), WO 2008/244380 (Bachand et. al), and US 2009/0068140(Bachand et. al). These references also contain significant disclosureregarding routes of administration, and other information regarding howto make, formulate, and use the compounds.

EXAMPLES

A table of abbreviations used in this Experimental section is set forthbelow.

-   -   DCM Dichloromethane    -   DMF N,N-dimethylformamide    -   HATU (O-7-azabenzotriazol-1-yl)-N, N, N′,N′-tetramethyluronium        hexafluorophosphate)    -   ES LC-MS Electrospray Liquid Chromatography Mass Spectrometry    -   THF Tetrahydrofuran    -   DIEA diisopropylethylamine    -   DMSO dimethylsulfoxide    -   DME dimethoxyethane    -   TEA Triethylamine    -   Pd(dppf)Cl₂        1,1′-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride        dichloromethane complex    -   Dess-Martin Dess-Martin periodinane    -   HRMS High Resolution Mass Spectroscopy

Intermediate 1: methyl{(1S)-1-1[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamate

Intermediate 1 can be prepared as illustrated by the reaction schemebelow.

A solution of1,1-dimethylethyl[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]carbamate(Intermediate 11) (3.8 g, 6.3 mmol) in DCM (40 mL) was treated with HCl(10 mL, 4M in dioxane) to give methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamate(Intermediate 1) as light yellow solid (3.5 g, quant.).

Intermediate 2:(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azasoiro[4.5]decane-3-carboxylicacid

Intermediate 2 can be prepared as illustrated in the reaction schemebelow.

To a stirred solution of methyl(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylate(Intermediate 14) (360 mg, 0.932 mmol) in a mixed solvents of THF (4mL), t-butanol (1 mL) and water (1 mL) was added LiOH (44 mg, 1.86mmol). The resulting mixture was stirred for 2 hrs at rt beforeacidified with 1N HCl to pH 3 and further diluted with ethyl acetate(100 mL). The solution was washed with brine. The organic layer wasdried over Na₂SO₄, filtered and evaporated to give(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid (Intermediate 2) (315 mg, yield: 91%) as solid. ES LC-MS m/z=373(M+H)⁺.

Intermediate 4: 2-amino-1-(4-bromophenyl)ethanone

To a stirred solution of 2-bromo-1-(4-bromophenyl)ethanone (130 g 0.478mol) in toluene (2500 mL) was added hexamethylenetetramine (65.6 g 0.478mol). The mixture was stirred at 40° C. for 16 hrs. The resulting solidwas filtered off and washed with toluene and ether to give a whitesolid. To a stirred suspension of this white solid in ethanol (800 mL)was added concentrated hydrochloride acid (300 mL). The mixture wasstirred at ambient temperature for 20 hrs. The solid was collected byfiltration and washed with ethanol and water and dried in vacuo to give2-amino-1-(4-bromophenyl)ethanone (4) (95 g, yield: 92%) as a whitesolid, which was used without purification for the next step. ¹H NMR(300 MHz, DMSO-d₆) δ ppm 8.58 (s, br, 2H), 7.96 (d, J=8.7 Hz, 2H), 7.81(d, J=8.7 Hz, 2H), 4.48-4.52 (m, 2H). ES LC-MS m/z=214, 216 (M+H)⁺.

Intermediate 5: 1,1-dimethylethyl[2-(4-bromophenyl)-2-oxoethyl]carbamate

To a mixture of 2-amino-1-(4-bromophenyl)ethanone hydrochloride(Intermediate 4) (50 g, 0.2 mol), Boc₂O (48 g, 0.22 mol) in DCM (1000mL) was added TEA (68.8 mL, 0.5 mol) dropwise at 0° C. After addition,the resulting mixture was stirred at ambient temperature overnight andwas filtered. The filtration was washed with 1 N HCl (300 mL×3) andbrine, dried over Na₂SO₄, concentrated in vacuo to give an off-whitesolid, which was further washed with petroleum ether to afford1,1-dimethylethyl[2-(4-bromophenyl)-2-oxoethyl]carbamate (Intermediate5) (40 g, yield: 64%). ¹H NMR (300 MHz, CDCl3) δ ppm 7.83 (d, J=8.7 Hz,2H), 7.65 (d, J=8.7 Hz, 2H), 5.48 (s, br, 1H), 4.60-4.62 (m, 2H), 1.49(s, 9H). ES LC-MS m/z=336 (M+Na)⁺.

Intermediate 6:1,1-dimethylethyl{2-oxo-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl}carbamate

Intermediate 6 can be prepared as illustrated in the reaction schemebelow.

Pd(dppf)Cl₂ (2.6 g 3.18 mmol) was added to a mixture of1,1-dimethylethyl[2-(4-bromophenyl)-2-oxoethyl]carbamate (Intermediate5) (20 g, 63.7 mmol), bis(pinacolato)diboron (19.4 g, 76.4 mmol) andKOAc (24.8 g, 0.254 mol) in dioxane (300 mL), the flask was purged withnitrogen (3×) and heated to 80° C. for 16 hrs under nitrogen atmosphere.The reaction mixture was diluted with hexane (300 mL), filtered,concentrated and the residue was purified by chromatography on silicagel (petroleum ether/ethyl acetate=5/1) to give1,1-dimethylethyl{2-oxo-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl}carbamate(Intermediate 6) (13.3 g, yield: 58%) as a white solid. ¹H NMR (300 MHz,CDCl3) δ ppm 7.90-7.93 (m, 4H), 5.55 (s, br, 1H), 4.68 (s, 2H), 1.48 (s,9H), 1.35 (s, 12H). ES LC-MS m/z=384 (M+Na)⁺.

Intermediate 7:1,1-dimethylethyl(2S)-2-({[2-(4-bromophenyl)-2-oxoethyl]amino}carbonyl)-1-pyrrolidinecarboxylate

A mixture of 1-{[(1,1-dimethylethyl)oxy]carbonyl}-L-proline (50 g, 0.233mol), HATU (106 g, 0.279 mol) and DIEA (150 mL) in DMF (400 mL) wasstirred at ambient temperature for 10 min.2-Amino-1-(4-bromophenyl)ethanone hydrochloride (Intermediate 4) (70 g,0.279 mol) in DMF (500 mL) was added and the resulting mixture wasstirred overnight before diluted with EtOAc (4 L). The solution waswashed with 1N HCl (500 mL×4) and brine, dried over Na₂SO₄,concentrated. The crude product was recrystallized from a mixture ofpetroleum ether/ethyl acetate (2/1) to give1,1-dimethylethyl(2S)-2-({[2-(4-bromophenyl)-2-oxoethyl]amino}carbonyl)-1-pyrrolidinecarboxylate(Intermediate 7) (58.4 g, yield: 61%) as yellow solid. ¹H NMR (300 MHz,DMSO) δ ppm 8.22 (s, 1H), 7.93 (d, J=8.4 Hz, 2H), 7.77 (d, J=8.4 Hz,2H), 4.46-4.51 (m, 2H), 4.15-4.21 (m, 1H), 3.28-3.40 (m, 2H), 1.78-1.90(m, 4H), 1.29-1.41 (m, 9H). ES LC-MS m/z=411.1, 4113.1 (M+H)⁺.

Intermediate 8:1,1-dimethylethyl(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinecarboxylate

A mixture of1,1-dimethylethyl(2S)-2-({[2-(4-bromophenyl)-2-oxoethyl]amino}carbonyl)-1-pyrrolidinecarboxylate(7) (40.0 g, 97.2 mmol) and NH₄OAc (60 g, 0.778 mol) in xylene (400 mL)was heated to 150° C. for 5 hrs in a sealed reactor. The reactionmixture was concentrated, and the residue was dissolved in EtOAc (500mL) and washed with aqueous NaHCO₃ and brine. The organic phase wasdried over Na₂SO₄, concentrated to dryness. The crude product waspurified by chromatography on silica gel (petroleum ether/ethylacetate=1/1) to give1,1-dimethylethyl(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinecarboxylate(Intermediate 8) (34 g, yield: 89%) as brown solid. ¹H NMR (300 MHz,CDCl₃) δ ppm 7.52 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.20 (s,1H), 5.58-5.71 (m, 1H), 3.38-3.42 (m, 1H), 2.80-2.87 (m, 2H), 2.03-2.06(m, 2H), 1.88-2.00 (m, 2H), 1.49 (s, 9H). ES LC-MS m/z=392, 394 (M+H)⁺.

Intermediate 9: 4-(4-bromophenyl)-2-[(2S)-2-pyrrolidinyl]-1H-imidazole

1,1-dimethylethyl(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinecarboxylate(Intermediate 8) (72.4 g, 185 mmol) was treated with saturated HCl indioxane (200 mL), and stirred at ambient temperature overnight. Theresulting solid was filtered and washed with petroleum ether to give4-(4-bromophenyl)-2-[(2S)-2-pyrrolidinyl]-1H-imidazole (Intermediate 9)(60 g, yield: 90%) as yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 8.10(s, 2H), 7.88 (d, J=6.6 Hz, 2H), 7.70 (d, J=6.6 Hz, 2H), 7.49 (s, 1H),7.32 (s, 1H), 7.16 (s, 1H), 4.50-4.52 (m, 1H), 3.15-3.40 (m, 2H),1.88-2.88 (m, 4H). ES LC-MS m/z=291.1, 293.1 (M+H)⁺.

Intermediate 10: methyl[(1S)-1-({(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

N-[(methyloxy)carbonyl]-L-valine (43.1 g, 0.246 mol) and HATU (93.5 g,0.246 mol) in DCM (1000 mL) was stirred for 10 min.4-(4-Bromophenyl)-2-[(2S)-2-pyrrolidinyl]-1H-imidazole (Intermediate 9)(60 g, 0.205 mol) was introduced, followed by DIEA (82.6 mL, 0.308 mol)dropwise. The mixture was stirred at ambient temperature overnightbefore diluted with DCM (1000 mL) and washed with aqueous NaHCO₃ andbrine. The organic phase was dried over Na₂SO₄, concentrated to dryness.The crude product was purified by chromatography on silica gel(petroleum ether/ethyl acetate=1/1) to give methyl[(1S)-1-({(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 10) (62 g, yield: 67%) as yellow solid. ¹H NMR (300 MHz,CDCl3) δ:7.43-7.51 (m, 4H), 7.17 (s, 1H), 5.53-5.57 (m, 1H), 5.20-5.22(m, 1H), 5.29-5.33 (m, 1H), 3.64-3.71 (m, 5H), 2.99-3.03 (m, 1H),1.88-2.31 (m, 4H), 0.88-0.92 (m, 6H). ES LC-MS m/z=449, 451 (M+H)⁺.

Intermediate 11:1,1-dimethylethyl[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]carbamate

To a mixture of methyl[(1S)-1-({(2S)-2-[4-(4-bromophenyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 10) (62 g, 0.138 mol), 1,1-dimethylethyl{2-oxo-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl}carbamate(Intermediate 6)

(47.7 g, 0.152 mol) and NaHCO₃ (34.2 g, 0.414 mol) in a mixed DME (800mL) and water (260 mL) was added Pd(dppf)Cl₂ (5.63 g, 6.9 mmol). Theflask was purged with nitrogen (3×) before heated to 80° C. for 16 hrs.The reaction was cooled down to rt and filtered. The filtrate wasdiluted with EtOAc (1000 mL), and the solution was washed with aqueousNaHCO₃ and brine, dried over Na₂SO₄, concentrated. The residue waspurified by chromatography on silica gel (petroleum ether/ethylacetate=1/2) to give1,1-dimethylethyl[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl]-4-biphenylyl)-2-oxoethyl]carbamate(Intermediate 11) (45 g, yield: 54%) as yellow solid. ¹H NMR (300 MHz,CDCl3) δ ppm 8.03 (d, J=8.4 Hz, 2H), 7.88-760 (m, 6H), 5.58 (S, br, 1H),5.42 (m, 1H), 5.28-5.30 (m, 1H), 4.71 (s, 2H), 4.32-4.35 (m, 1H),3.70-3.84 (m, 5H), 2.96 (s, br, 1H), 1.96-2.11 (m, 4H), 1.49 (s, 9H),0.88-0.92 (m, 6H). ES LC-MS m/z=604, (M+H)⁺;

Intermediate 12: methylN-[(methyloxy)carbonyl]-L-valyl-(4R)-4-hydroxy-L-prolinate

To a stirred solution of N-[(methyloxy)carbonyl]-L-valine (2.89 g, 16.52mmol) in DCM were added TEA (3.51 g, 34.7 mmol) and HATU (3 g, 16.52mmol). After approximately 10 min stirring, methyl(4R)-4-hydroxy-L-prolinate hydrochloride (3 g, 16.52 mmol) wasintroduced. The resulting mixture was stirred for additional 4 hrs at rtbefore quenched with NaHCO₃ (ss). The layers were separated and theaqueous layer was extracted with DCM (2×). The combined organic phasewas dried over Na₂SO₄, filtered and concentrated. The crude product waspurified by column chromatography (silica gel, 0 to 70′)/0 ethyl acetatein hexane) to give methylN-[(methyloxy)carbonyl]-L-valyl-(4R)-4-hydroxy-L-prolinate (Intermediate12) (3.5 g, yield: 70%). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.34-5.54(m, 1H) 4.69 (t, J=8.41 Hz, 1H) 4.55 (br. s., 1H) 4.21 (s, 1H) 3.92-4.08(m, 1H) 3.74 (s, 4H) 3.66 (s, 3H) 2.29-2.50 (m, 1H) 1.92-2.19 (m, 2H)0.86-1.13 (m, 6H). ES LC-MS m/z=303.5 (M+H)⁺.

Intermediate 13: methylN-[(methyloxy)carbonyl]-L-valyl-4-oxo-L-prolinate

To a stirred solution ofN-[(methyloxy)carbonyl]-L-valyl-(4R)-4-hydroxy-L-prolinate (Intermediate12) (3.5 g, 11.9 mmol) in DCM (80 mL) was added Dess-Martin (10 g) at rtThe resulting mixture was stirred for additional 4 hours before quenchedwith 5% aq. sodium thiosulfate (350 mL) and then sat. NaHCO₃ (200 mL).Stirred was continued for 10 min and the mixture was extracted with DCM(2×300 mL). The organic layer was dried over MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography(silica gel, 0 to 70% ethyl acetate in hexane) to give methylN-[(methyloxy)carbonyl]-L-valyl-4-oxo-L-prolinate (Intermediate 13) (701mg, yield: 20%). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.25 (d, J=9.18Hz, 1H) 5.07 (dd, J=10.74, 2.93 Hz, 1H) 4.36 (d, J=17.77 Hz, 1H)3.97-4.23 (m, 2H) 3.69-3.77 (m, 3H) 3.63 (s, 3H) 2.92 (dd, J=18.94,10.74 Hz, 1H) 2.62 (dd, J=18.94, 2.73 Hz, 1H) 2.01 (t, J=3.32 Hz, 1H)0.86-1.16 (m, 6H).

Intermediate 14:methyl(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2azaspiro[4.5]decane-3-carboxylate

Methyl N-[(methyloxy)carbonyl]-L-valyl-4-oxo-L-prolinate (Intermediate13) (650 mg, 2.164 mmol), (2S,4S)-2,4-pentanediol (902 mg, 8.66 mmol)and TsOH (82 mg, 0.43 mmol) were heated to reflux in toluene (40 mL)with Dean Stark trap overnight. After cooled down to rt and diluted withethyl acetate, the resulting solution was washed with NaHCO₃ (ss) andbrine. The organic later was dried over MgSO4, filtered and evaporated.The crude product was purified by column chromatography (silica gel, 0to 50% ethyl acetate in hexane) to give methyl(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylate(Intermediate 14) (366 mg, yield: 44%) as an oil. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 5.38-5.51 (m, 1H) 4.54 (t, J=8.03 Hz, 1H) 4.25-4.36(m, 1H) 3.91-4.05 (m, 2H) 3.85 (d, J=10.04 Hz, 1H) 3.54-3.77 (m, 6H)2.46-2.53 (m, 1H) 2.12 (dd, J=12.92, 7.65 Hz, 1H) 2.00-2.06 (m, 1H)1.54-1.75 (m, 3H) 1.10-1.25 (m, 6H) 1.03 (d, J=6.78 Hz, 3H) 0.84-0.96(m, 3H). ES LC-MS m/z=409.3 (M+Na)⁺.

Intermediates 15 and 17 were prepared using procedures similar to theprocedures outlined in the preparation of Intermediate 2:

Intermediate 15:(3S,7R,9R)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.53 (br. s., 1H), 7.49 (d, 1H), 4.34(d, 1H), 4.24 (t, 1H), 4.06 (br. m, 2H), 3.93 (t, 1H), 3.37 (m, 4H),2.35 (m, 1H), 2.09-1.86 (br. m, 2H), 1.59 (m, 2H), 1.17 (m, 6H), 0.91(m, 6H).

Intermediate 16:methyl(8S)-7-{N-[(methyloxy)carbonyl]-L-valyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylate

ES LC-MS m/z=345.3 (M+H)⁺.

Intermediate 17:(8S)-7-{N-[(methyloxy)carbonyl]-L-valyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.56 (br. s., 1H) 7.40 (d, J=8.39 Hz,1H) 4.33 (dd, J=8.78, 7.02 Hz, 1H) 3.81-4.10 (m, 5H) 3.41-3.66 (m, 5H)2.28-2.43 (m, 1H) 1.94-2.11 (m, 1H) 1.74-1.94 (m, 1H) 0.67-1.05 (m, 6H).ES LC-MS m/z=331.6 (M+H)⁺.

Example 1 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S,7S,9S)-7,9-dimethyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

To a stirred solution of((3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid (Intermediate 2) (96 mg, 0.258 mmol) in DMF (2 mL) were added TEA(78 mg, 0.773 mmol) and HATU (108 mg, 0.284 mmol). After ˜3 minstirring, methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (Intermediate 1) (149 mg, 0.258 mmol) was introduced.After the reaction was stirred for additional 2 hrs at rt, the mixturewas directly loaded to RP HPLC, eluting with 5 to 80% acetonitrile/water(0.2% NH₃H₂O (conc.)) to give methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S,7S,9S)-7,9-dimethyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 3) as solid (135 mg, yield: 61%). ¹HNMR (400 MHz,CHLOROFORM-d) δ ppm 10.40-11.01 (m, 1H) 7.32-8.30 (m, 10H) 5.28 (br. s.,2H) 4.53-4.96 (m, 4H) 4.17-4.53 (m, 1H) 3.40-4.17 (m, 11H) 2.83-3.22 (m,1H) 2.26-2.74 (m, 3H) 1.54-2.26 (m, 8H) 0.47-1.43 (m, 18H). ES LC-MSm/z=858.6 (M+H)⁺.

To a stirred solution of methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S,7S,9S)-7,9-dimethyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 3) (135 mg, 0.157 mmol) in dioxane (3 mL) was addedammonium acetate (121 mg, 1.57 mmol). The reaction mixture was heated to110° C. in a sealed tube overnight. Cooled down to rt, filtered offexcess of ammonium acetate. The filtrate was evaporated and the residuewas purified by column chromatography (silica gel, 0-15% methanol inethyl acetate) to give methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S,7S,9S)-7,9-dimethyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Example 1) as solid (81 mg, yield: 58%). ¹HNMR (400 MHz, CHLOROFORM-d)δ ppm 10.23-11.01 (m, 1H) 7.31-8.08 (m, 8H) 7.23 (d, J=8.03 Hz, 2H)5.13-5.89 (m, 4H) 3.34-4.69 (m, 13H) 2.84-3.31 (m, 2H) 2.63-2.84 (m, 1H)2.29-2.53 (m, 1H) 1.85-2.29 (m, 4H) 1.56-1.85 (m, 4H) 1.16-1.47 (m, 6H)0.63-1.16 (m, 12H). HRMS: (M+H)⁺ calcd: 835.4456. found: 835.4458.

Example 2 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

Methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamatewas obtained from(8S)-7-{N-[(methyloxy)carbonyl]-L-valyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylicacid (Intermediate 17) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (Intermediate 1), following the similar two-stepsynthetic procedures outlined in Example 1. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 10.00-11.36 (m, 2H) 7.56 (br. s., 10H) 7.02-7.34 (m,2H) 5.05-5.89 (m, 4H) 3.76-4.65 (m, 6H) 3.53-3.83 (m, 6H) 2.77-3.54 (m,2H) 2.26-2.70 (m, 2H) 1.45-2.26 (m, 6H) 0.61-1.25 (m, 12H). HRMS: (M+H)⁺calcd: 797.3986. found: 797.3981.

Example 3 dimethyl(4,4′-biphenyldiylbis{1H-imidazole-4,2-diyl[(3S,7S,9S)-7,9-dimethyl-6,10-dioxa-2-azaspiro[4.5]decane-3,2-diyl][(2S)-3-methyl-1-oxo-1,2-butanediyl]})biscarbamate

Under N₂ atmosphere, to a stirred suspension of1,1′-(4,4′-biphenyldiyl)bis(2-bromoethanone) (113 mg, 0.285 mmol,prepared according to the procedures provided in WO2009020825) inacetonitrile (5 mL) was added(3S,7S,9S)-7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid (Intermediate 2) (212 mg, 0.571 mg), followed by addition of TEA(57.5 mg, 0.571 mmol). The mixture was stirred at 50° C. until thesuspension became clear. After it was cooled down to rt, the reactionmixture was diluted with ethyl acetate and washed with brine. Theorganic phase was dried over MgSO₄, filtered and evaporated to give4,4′-biphenyldiylbis-2-oxo-2,1-ethanediyl(3S,7S,9S,3′S,7′S,9′S)bis(7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylate)(Intermediate 18) (280 mg, quant.). ES LC-MS m/z=979.6 (M+H)⁺.

To a stirred solution of 4,4′-biphenyldiylbis-2-oxo-2,1-ethanediyl(3S,7S,9S,3′S,7′S,9′S)bis(7,9-dimethyl-2-{N-[(methyloxy)carbonyl]-L-valyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylate)(Intermediate 18) (280 mg, 0.286 mmol) in dioxane (5 mL) in a sealedtube was added ammonium acetate (441 mg, 5.72 mmol). The reactionmixture was heated to 110° C. overnight. Cooled down to rt, filtered offexcess of ammonium acetate. The filtrate was evaporated and the residuewas purified by column (silica gel, 0-15% methanol in ethyl acetate) togive dimethyl(4,4′-biphenyldiylbis{1H-imidazole-4,2-diyl[(3S,7S,9S)-7,9-dimethyl-6,10-dioxa-2-azaspiro[4.5]decane-3,2-diyl][(2S)-3-methyl-1-oxo-1,2-butanediyl]})biscarbamate(Example 3) as a solid (112 mg, yield: 40%). ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 10.30-10.99 (m, 2H) 7.68-8.00 (m, 2H) 7.61 (d,J=7.53 Hz, 6H) 7.26-7.35 (m, 4H) 5.59 (d, J=8.53 Hz, 1H) 5.23-5.39 (m,1H) 4.44 (dd, J=8.41, 5.14 Hz, 2H) 4.02-4.23 (m, 4H) 3.97 (d, J=10.29Hz, 2H) 3.72 (s, 6H) 3.62 (d, J=10.29 Hz, 2H) 3.04-3.32 (m, 2H) 2.70 (d,J=13.05 Hz, 2H) 1.93 (br. s., 2H) 1.56-1.82 (m, 4H) 1.13-1.46 (m, 12H)0.60-1.02 (m, 12H). HRMS: (M+H)⁺ calcd: 939.4980. found: 939.4981.

Example 4 dimethyl(4,4′-biphenyldiylbis{1H-imidazole-4,2-diyl(8S)-1,4-dioxa-7-azaspiro[4.4]nonane-8,7-diyl[(2S)-3-methyl-1-oxo-1,2-butanediyl]})biscarbamate

Dimethyl(4,4′-biphenyldiylbis{1H-imidazole-4,2-diyl(8S)-1,4-dioxa-7-azaspiro[4.4]nonane-8,7-diyl[(2S)-3-methyl-1-oxo-1,2-butanediyl]})biscarbamatewas obtained from 1,1′-(4,4′-biphenyldiyl)bis(2-bromoethanone) and(8S)-7-{N-[(methyloxy)carbonyl]-L-valyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylicacid (Intermediate 17) using a process similar to the two-stepprocedures outlined in Example 3. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm11.10-11.23 (m, 1H) 10.10-11.63 (m, 2H) 7.40-8.15 (m, 8H) 7.27-7.40 (m,2H) 5.23-5.88 (m, 2H) 4.31 (dd, J=8.68, 6.54 Hz, 2H) 3.83-4.21 (m, 10H)3.57-3.83 (m, 6H) 3.05-3.46 (m, 3H) 2.47 (dd, J=13.46, 8.59 Hz, 2H)1.51-2.20 (m, 2H) 1.08 (d, J=6.83 Hz, 2H) 0.60-0.97 (m, 12H). HRMS:(M+H)⁺ calcd: 855.4041. found: 855.4039.

Example 5 methyl((1S)-1-methyl-2-{(3S)-3-[4-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-6,10-dioxa-2-azaspiro[4.5]dec-2-yl}-2-oxoethyl)carbamate

To a stirred solution of N-[(methyloxy)carbonyl]-L-alanine (22.5 mg,0.153 mmol, prepared according to the procedure provided inWO2003055474) in DMF (2 mL) were added TEA (15.5 mg, 0.153 mmol) andHATU (58.2 mg, 0.153 mmol). After ˜3 min stirring, methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Example 19) (100 mg, 0.153 mmol) was introduced. After stirred foradditional 2 hrs at rt, the reaction mixture was directly loaded to RPHPLC, eluting with 5 to 80% acetonitrile/water (0.2% NH₃H₂O(conc.)), togive methyl((1S)-1-methyl-2-{(3S)-3-[4-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-6,10-dioxa-2-azaspiro[4.5]dec-2-yl}-2-oxoethyl)carbamate(Example 5) as solid (36 mg, yield: 29%). ¹H NMR (400 MHz, CHLOROFORM-d)δ ppm 10.04-11.11 (m, 2H) 7.37-8.02 (m, 8H) 7.18-7.36 (m, 2H) 5.60 (br.s., 2H) 5.17-5.40 (m, 2H) 3.17-4.73 (m, 14H) 2.79-3.19 (m, 1H) 2.45-2.81(m, 1H) 2.29-2.45 (m, 1H) 1.49-2.29 (m, 8H) 1.22-1.47 (m, 3H) 0.73-1.16(m, 6H). HRMS: (M+H)⁺ calcd: 783.3830. found: 783.3832.

Intermediate 19: Preparation of methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

Intermediate 19 can be prepared as illustrated in the reaction schemebelow.

Intermediate 21:phenylmethyl(3S)-3-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate

To a stirred solution of(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid (Intermediate 20) (878 mg, 2.73 mmol) in DMF (10 mL) were added TEA(829 mg, 8.20 mmol) and HATU (1039 mg, 2.73 mmol). After ˜3 minstirring, methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 1) (1575 mg, 2.73 mmol) was added. The resulting mixturewas stirred for additional 2 hrs at rt before quenched with NaHCO3 (ss)and extracted with EtOAc (3×). The combined organic layers was driedover Na₂SO₄, filtered and evaporated. The crude product was purified bycolumn chromatography (silica gel, 0 to 6% MeOH (2M ammonia) in DCM) togive phenylmethyl(3S)-3-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate(Intermediate 21) as a solid (1.76 g, yield: 80%). ES LC-MS m/z=807.5(M+H)⁺.

Intermediate 22: phenylmethyl(3S)-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate

To a stirred solution of(3S)-3-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate(Intermediate 21) (1.76 g, 2.18 mmol) in dioxane (5 mL) in a sealed tubewas added ammonium acetate (1.68d, 21.8 mmol). The reaction mixture washeated to 110° C. overnight. Cooled down to rt, filtered off excess ofammonium acetate. The filtrate was evaporated and the residue waspurified by column (silica gel, 0-15% methanol in ethyl acetate) to givephenylmethyl(3S)-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate(Intermediate 22) (1.44 g, yield 84%) as foam. ES LC-MS m/z=788.5(M+H)⁺.

Intermediate 19: methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

Phenylmethyl(3S)-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-6,10-dioxa-2-azaspiro[4.5]decane-2-carboxylate(Intermediate 22) (1.44 g, 1.83 mmol) was hydrogenated in ethanol (100mL) with balloon under the catalysis of Pd/C for 20 hrs to give methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 19). ES LC-MS m/z=654.4 (M+H)⁺.

Intermediate 20:(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid

Intermediate 20 can be prepared as illustrated in the reaction schemebelow.

Intermediate 23: 2-methyl 1-(phenylmethyl)(2S)-4-oxo-1,2-pyrrolidinedicarboxylate

At −78° C., to a stirred solution of oxalyl chloride (5.97 g, 47 mmol)in DCM (200 mL) was slowly added DMSO (4.8 g, 61.5 mmol). After 10 minstirring, a solution of 2-methyl 1-(phenylmethyl)(2S,4R)-4-hydroxy-1,2-pyrrolidinedicarboxylate (10.1 g, 36.2 mmol) inDCM (30 mL) was annulled into the reaction flask. Stirring was continuedfor 60 min before addition of triethylamine (10.98 g, 108 mmol). Coolingbath was then removed and the reaction mixture was allowed to slowlywarm up to 0° C., and quenched with sat. NH₄CI solution. The layers wereseparated and the aqueous layer was extracted with DCM (2×). Thecombined organic layers was dried over MgSO₄, filtered and evaporatedThe crude product was purified by column chromatography (silica gel 0 to50% ethyl acetate in hexane) to give 2-methyl 1-(phenylmethyl)(2S)-4-oxo-1,2-pyrrolidine dicarboxylate (Intermediate 23) (6.3 g,yield: 63%). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.29-7.56 (m, 5H)5.08-5.37 (m, 2H) 4.80-5.02 (m, 1H) 3.89-4.08 (m, 2H) 3.53-3.88 (m, 3H)2.95 (dd, J=18.82, 10.79 Hz, 1H) 2.63 (dd, 1H).

Intermediate 24: 3-methyl 2-(phenylmethyl)(3S)-6,10-dioxa-2-azaspiro[4.5]decane-2,3-dicarboxylate

3-Methyl 2-(phenylmethyl)(3S)-6,10-dioxa-2-azaspiro[4.5]decane-2,3-dicarboxylate (Intermediate24) (1.02 g, yield 84%) was prepared from 2-methyl 1-(phenylmethyl)(2S)-4-oxo-1,2-pyrrolidinedicarboxylate (1.0 g, 3.61 mmol) and1,3-propanediol (0.55 g, 7.21 mmol) following the similar procedureoutlined in the preparation of Intermediate 14 ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 7.05-7.61 (m, 5H) 4.91-5.25 (m, 2H) 4.29-4.68 (m,1H) 3.75-4.07 (m, 5H) 3.38-3.75 (m, 4H) 2.25-2.67 (m, 2H) 1.51-2.00 (m,1H). ES LC-MS m/z=336.6 (M+H)⁺.

Intermediate 20:(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid

(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid (Intermediate 20) (878 mg, yield: 90%) was obtained from 3-methyl2-(phenylmethyl) (3S)-6,10-dioxa-2-azaspiro[4.5]decane-2,3-dicarboxylate(Intermediate 24) (1.02 g, 3.04 mmol) and LiOH (80 mg, 3.35 mmol),following the similar procedure outlined in the preparation ofintermediate 2. ES LC-MS m/z=322.2 (M+H)⁺.

Intermediates 26, 27, 28, 29, 31, 33 and 34 were prepared usingprocedures similar to those described in the preparation of Intermediate20:

Intermediate 25: 3-methyl 2-(phenylmethyl)(3S)-6,10-dioxa-2-azaspiro[4.5]decane-2,3-dicarboxylate-d₂

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.27-7.46 (m, 5H) 4.97-5.27 (m, 2H)4.29-4.64 (m, 1H) 3.72-4.02 (m, 6H) 3.49-3.72 (m, 3H) 2.19-2.67 (m, 2H).ES LC-MS m/z=338.2 (M+H)⁺.

Intermediate 26:(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid-d₂

ES LC-MS m/z=324.2 (M+H)⁺.

Intermediate 27:(3S)-8,8-dimethyl-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid

Intermediate 28:(3S)-2-{[(phenylmethyl)oxy]carbonyl}-6,10-dioxa-2-azaspiro[4.5]decane-3-carboxylicacid-d₆

Intermediate 29:(8S)-7-{[(phenylmethyl)oxy]carbonyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylicacid-d₄

Intermediate 30: 8-methyl 7-(phenylmethyl)(2R,3R,8S)-2,3-dimethyl-1,4-dioxa-7-azaspiro[4.4]nonane-7,8-dicarboxylate

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.12-1.24 (m, 6H), 2.00-2.18 (m, 1H),2.35-2.46 (m, 1H), 3.32 (s, 1H), 3.35-3.44 (m, 1H), 3.56 (s, 2H), 3.64(s, 3H), 4.28-4.37 (m, 1H), 4.38-4.45 (m, 0H), 5.09 (s, 2H), 7.24-7.41(m, 5H) LC-MS (ESI): m/z=350.1 (M+H)⁺;

Intermediate 31:(2R,3R,8S)-2,3-dimethyl-7-{[(phenylmethyl)oxy]carbonyl}-1,4-dioxa-7-azaspiro[4.4]nonane-8-carboxylicacid

LC-MS (ESI): m/z=336.2 (M+H)⁺;

Intermediate 32: 8-methyl 7-(phenylmethyl)(2S,3S,8S)-2,3-dimethyl-1,4-dioxa-7-azaspiro[4.4]nonane-7,8-dicarboxylate

¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 1.18-1.31 (m, 6H), 2.23 (dd, J=13.1,5.9 Hz, 1H), 2.39 (dt, J=13.1, 8.0 Hz, 1H), 3.49-3.66 (m, 6H), 3.76 (s,1H), 4.39-4.56 (m, 1H), 4.98-5.26 (m, 2H), 7.36 (s, 5H) LC-MS (ESI):m/z=350.1 (M+H)⁺;

Intermediate 33:(2S,3S,8S)-2,3-dimethyl-7-{[(phenylmethyl)oxy]carbonyl}-1,4-dioxa-7-azasoiro[4.4]nonane-8-carboxylicacid

LC-MS (ESI): m/z=336.2 (M+H)⁺;

Intermediate 34:(5'S)-1′-{[(phenylmethyl)oxy]carbonyl}tetrahydrospiro[furo[3,4-d][1,3]dioxole-2,3′-pyrrolidine]-5′-carboxylicacid

LC-MS (ESI): m/z=350.2 (M+H)⁺;

Example 6 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(3S)-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azasoiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

Methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(3S)-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(47 mg, yield: 53%) was prepared from methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 19) (68 mg, 0.104 mmol), N-[(methyloxy)carbonyl]-L-valine(18.2 mg, 0.104) and HATU (40 mg, 0.104 mmol), following the similarprocedure outlined in Example 5. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm10.20-11.06 (m, 2H) 7.35-8.02 (m, 8H) 7.22 (d, J=7.78 Hz, 2H) 4.97-5.89(m, 4H) 3.22-4.58 (m, 15H) 2.83-3.20 (m, 1H) 2.58-2.83 (m, 1H) 2.29-2.58(m, 1H) 1.57-2.31 (m, 8H) 0.64-1.22 (m, 12H). HRMS: (M+H)⁺ calcd:811.4143. found: 811.4142.

Examples 7 to 11 were prepared using the similar synthetic sequencedescribed for synthesis of Example 5

Example 7 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(3S)-8,8-dimethyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 11.84 (br. s., 1H), 11.80 (br. s, 1H),7.90-7.60 (br. m, 9H), 7.52 (s, 1H), 7.36-7.25 (br. m, 2H), 5.09 (br. m,1H), 4.98 (m, 1H), 4.49 (br. m, 1H), 4.14-4.02 (br. m, 2H), 3.81 (br,2H), 3.73-3.40 (br. m, 11H), 2.65 (m, 1H), 2.43-2.09 (br. m, 3H),2.07-1.81 (br. m, 4H), 1.05-0.75 (br. m, 18H) ES LC-MS m/z=839 (M+H)⁺Purity (LC/MS) 96%

Example 8 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(3S)-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6,10-dioxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate-d₆

¹H NMR (400 MHz, CD₃OD) δ ppm 7.62 (m, 8H), 7.29 (m, 2H), 5.17 (m, 2H),4.20 (m, 2H), 4.00 (m, 2H), 3.88 (m, 2H), 3.65 (s, 6H), 2.65-2.03 (m,8H), 0.90 (m, 12H).

LC-MS for C₄₃H₄₈N₈O₈D₆ (M+H)⁺ calc: 817. found: 817.

HRMS for C₄₃H₄₈N₈O₈D₆ (M+H)⁺ calc: 817.4519. found: 817.4517

Purity (LC/MS) 95%

Example 9 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate-d₄

¹H NMR (400 MHz, CD₃OD) δ ppm 7.60 (m, 8H), 7.28 (m, 2H), 5.17 (m, 2H),4.23 (m, 1H), 4.15 (m, 1H), 4.06 (m, 1H), 3.97 (m, 1H), 3.88 (m, 2H),3.64 (s, 6H), 2.50-2.00 (m, 8H), 0.90 (m, 12H). LC-MS for C₄₂H₄₈N₈O₈D₄(M+H)⁺ calc: 801. found: 801.

HRMS for C₄₂H₄₈N₈O₈D₄ (M+H)⁺ calc: 801.4237. found: 801.4238

Purity (LC/MS) 91%

Example 10 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(2R,3R,8S)-2,3-dimethyl-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-5-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.85 (d, J=6.4 Hz, 12H), 1.24 (dd,J=11.9, 5.8 Hz, 6H), 1.87-2.06 (m, 4H), 2.08-2.21 (m, 2H), 2.34-2.43 (m,1H), 3.54 (s, 6H), 3.59-3.68 (m, 2H), 3.69-3.75 (m, 2H), 3.81 (br. s.,2H), 3.97-4.14 (m, 3H), 5.00-5.17 (m, 2H), 7.25-7.33 (m, 2H), 7.49-7.54(m, 2H), 7.62-7.71 (m, 4H), 7.78 (d, J=7.7 Hz, 4H), 11.79 (br. s., 2H);LC-MS (ESI): m/z=825.5 (M+H)⁺; HRMS: (M+H)⁺ calcd, 825.4299. found,825.4302. Purity: 92%

Example 11 methyl [(1S)-1-({(2S)-2-[4-(4′-{2-[(2S,3S, 8S)-2,3-dimethyl-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-5-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.70-0.96 (m, 7H), 1.12-1.35 (m, 4H),1.85-2.04 (m, 3H), 2.04-2.25 (m, 2H), 2.31-2.47 (m, 1H), 3.33 (s, 7H),3.45-3.61 (m, 8H), 3.63-3.89 (m, 7H), 3.94-4.15 (m, 4H), 4.91-5.17 (m,2H), 7.20-7.40 (m, 2H), 7.49-7.59 (m, 1H), 7.60-7.70 (m, 2H), 7.70-7.81(m, 2H), 7.81-7.93 (m, 2H), 7.96-8.08 (m, 0H), 11.59-12.00 (m, 2H);LC-MS (ESI): m/z 825.5 (M+H)⁺; HRMS: (M+H)⁺ calcd, 825.4299. found,825.4302.

Intermediate 36:(8S)-7-{[(phenylmethyl)oxy]carbonyl}-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxylicacid

Intermediate 35: 8-methyl 7-(phenylmethyl)(8S)-1,4-dithia-7-azaspiro[4.4]nonane-7,8-dicarboxylate

To a stirred solution of 2-methyl 1-(phenylmethyl)(2S)-4-oxo-1,2-pyrrolidinedicarboxylate (Intermediate 23) (680 mg, 2.452mmol) in anhydrous DCM (50 mL) was added 1,2-ethanethiodiol (462 mg, 4.9mmol) and followed by addition of BF₃ etherate (139 mg, 0.4 mmol). Theresulting mixture was stirred overnight at rt. before quenched withNaHCO₃ (ss). Layers were separated and the organic layer was dried,filtered and evaporated. The residue was purified by columnchromatography (silica gel, 0 to 50% ethyl acetate in hexane) to give8-methyl 7-(phenylmethyl)(8S)-1,4-dithia-7-azaspiro[4.4]nonane-7,8-dicarboxylate (Intermediate35) as an oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.48 (m, 5H)4.95-5.35 (m, 2H) 4.34-4.63 (m, 1H) 3.90-4.11 (m, 1H) 3.82-3.90 (m, 1H)3.78 (s, 2H) 3.50-3.69 (m, 1H) 3.15-3.45 (m, 4H) 2.74 (ddd, J=13.30,7.78, 1.25 Hz, 1H) 2.40-2.64 (m, 1H). ES LC-MS m/z=354.2 (M+H)

Intermediate 36:(8S)-7-{1{[(phenylmethyl)oxy]carbonyl}-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxylicacid

(8S)-7-{[(Phenylmethyl)oxy]carbonyl}-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxylic(Intermediate 36) acid (36) (640 mg, yield: 84%) was obtained from8-methyl 7-(phenylmethyl)(8S)-1,4-dithia-7-azaspiro[4.4]nonane-7,8-dicarboxylate (798 mg, 2.25mmol) and LiOH (60 mg, 2.5 mmol), following the procedure outlined inthe preparation of intermediate 2. ES LC-MS m/z=340.1 (M+H).

Intermediate 39: Preparation of methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

Intermediate 37:phenylmethyl(8S)-8-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate

Phenylmethyl(8S)-8-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl]-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate(Intermediate 37) (584 mg, yield 68%) was obtained from methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (Intermediate 1) (603 mg, 1.046 mmol),(8S)-7-{[(phenylmethyl)oxy]carbonyl}-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxylicacid (Intermediate 36) (355 mg, 1.046 mmol) and HATU (498 mg, 1.046mmol), following similar procedure outlined in preparation ofintermediate 21. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.88-8.03 (m, 2H)7.45-7.85 (m, 6H) 7.05-7.45 (m, 6H) 5.61 (br. s., 1H) 4.98-5.41 (m, 2H)4.42-4.92 (m, 2H) 4.35 (t, J=7.78 Hz, 1H) 3.93-4.06 (m, 1H) 3.86 (d,J=8.78 Hz, 3H) 3.62-3.80 (m, 4H) 3.35 (d, J=5.02 Hz, 4H) 3.21 (d, J=7.28Hz, 1H) 2.61-2.96 (m, 2H) 2.24 (br. s., 2H) 1.93-2.16 (m, 2H) 0.98-1.17(m, 3H) 0.91 (t, J=7.15 Hz, 6H). ES LC-MS m/z=825.2 (M+H).

Intermediate 38: Phenylmethyl(8S)-8-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate

Phenylmethyl(8S)-8-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate(Intermediate 38) (497 mg, 87%) was obtained from phenylmethyl(85)-8-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl]-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate(Intermediate 37) (584 mg, 0.71 mmol) and ammonium acetate (546 mg, 7.1mmol), following the similar procedure outlined in the preparation ofIntermediate (22). ES LC-MS m/z=806.4 (M+H).

Intermediate 39: methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

At rt, to a stirred solution of phenylmethyl(8S)-8-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate(Intermediate 38) (497 mg, 0.617 mmol) in TFA (6 mL) was added triflicacid (278 mg, 1.85 mmol). The resulting mixture was stirred at rt for 3hrs. Evaporated solvents, neutralized with NaHCO₃(SS) and the aqueousphase was extracted with DCM (15% IPA) (3×). The combined organic phaseswas dried, filtered and evaporated to give methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 39) (400 mg, yield: 97%).

ES LC-MS m/z=672.2 (M+H).

Example 12 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

Methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(88 mg, yield: 27%) was obtained from methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 39) (250 mg, 0.372 mmol), N-[(methyloxy)carbonyl]-L-valine(65 mg, 0.372 mmol) and HATU (141 mg, 0.372 mmol), following the similarprocedure outlined in Example 5. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm10.07-11.08 (m, 2H) 7.82 (br. s., 3H) 7.58 (d, J=6.02 Hz, 5H) 7.10-7.30(m, 2H) 5.54 (d, J=9.29 Hz, 2H) 5.17-5.42 (m, 3H) 4.01-4.48 (m, 2H)3.23-4.03 (m, 10H) 2.70-3.19 (m, 2H) 2.30-2.49 (m, 1H) 1.91-2.29 (m, 4H)1.19-1.56 (m, 4H) 1.07 (dd, J=10.54, 7.03 Hz, 2H) 0.58-0.97 (m, 10H).HRMS: (M+H)⁺ calcd: 829.3530. found: 829.3534.

Example 13methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

Methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(35 mg, yield: 27%) was obtained from methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 39) (100 mg, 0.149 mmol),(2S)-2-{[(methyloxy)carbonyl]amino}butanoic acid (24 mg, 0.15 mmol) andHATU (56.5 mg, 0.149 mmol), following the similar procedure outlined inExample 5. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.99-11.64 (m, 2H) 7.53(br. s., 8H) 7.12-7.34 (m, 2H) 5.26 (br. s., 2H) 3.18-4.64 (m, 17H) 2.79(br. s., 2H) 1.43-2.62 (m, 8H) 0.70-1.19 (m, 9H). HRMS: (M+H)⁺ calcd:815.3373. found: 815.3373.

Example 14 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-({[(methyloxy)carbonyl]amino}acetyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

Methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-({[(methyloxy)carbonyl]amino}acetyl)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(14 mg, yield: 24%) was obtained from methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(8S)-1,4-dithia-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate(Intermediate 39) (50 mg, 0.074 mmol), N-[(methyloxy)carbonyl]glycine(10 mg, 0.074 mmol) and HATU (28.3 mg, 0.074 mmol), following thesimilar procedure outlined in Example 5. ¹H NMR (400 MHz, CHLOROFORM-d)δ ppm 9.86-11.31 (m, 2H) 7.50 (br. s., 8H) 6.91-7.27 (m, 2H) 5.41-6.31(m, 2H) 5.24 (br. s., 2H) 3.12-4.45 (m, 18H) 2.77 (br. s., 2H) 1.77-2.50(m, 4H) 0.58-1.15 (m, 6H). HRMS: (M+H)⁺ calcd: 787.3055. found:787.3056.

Example 15 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

A solution of methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-2-azaspiro[4.5]dec-3-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate(Intermediate 104) (131 mg, 0.16 mmol) and ammonium acetate (122 mg, 1.6mmol) in dioxane (2 mL) was degassed and heated to 110° C. in a sealedtube for 18 h. The reaction was cooled to room temperature and dilutedwith ethyl acetate and filtered and concentrated in vacuo. The residuewas purified by C₁₈ RP chromatography eluting with 10-90%water/acetonitrile/0.2% NH₄OH, to afford an off-white solid (44 mg, 34%yield). ¹H NMR (400 MHz, METHANOL-d₄) 6 ppm 7.55-7.87 (m, 8H) 7.32 (br.s., 4H) 5.02-5.23 (m, 3H) 4.13-4.34 (m, 4H) 3.93-4.04 (m, 1H) 3.88 (br.s., 2H) 3.49-3.84 (m, 8H) 1.86-2.54 (m, 10H) 1.45-1.84 (m, 4H) 0.69-1.12(m, 12H). HRMS for C₄₄H₅₇N₈O₇ (M+H)⁺ calc: 809.4350. found: 809.4346.Purity (LC-MS): 96%.

Preparation of Intermediate 104

Intermediate 101: ethyl 8-oxa-2-azaspiro[4.5]decane-3-carboxylate

Intermediate 101 was obtained in quantitative yield as a racemate fromtetrahydro-2H-pyran-4-carbaldehyde (1.0 g, 8.8 mmol) following theprocedure outlined in WO 98/08850 pp. 50.

Intermediate 102: ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylate

To a solution of ethyl 8-oxa-2-azaspiro[4.5]decane-3-carboxylate (101)(200 mg, 0.94 mmol), HATU (392 mg, 1.03 mmol) andN-[(methyloxy)carbonyl]-L-valine (181 mg, 1.03 mmol) in anhydrous CH₂Cl₂(6 mL) was added Hunig's base (0.33 mL, 1.88 mmol) and the solutionstirred at rt under nitrogen. After 2 h, the reaction was concentratedin vacuo, purified by C₁₈ RP chromatography, eluting with 10-90%ACN/water/0.2% NH₄OH, to afford the product as a yellow oil (313 mg, 90%yield).

Intermediate 103:2-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylicacid

To a solution of ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylate(102) (310 mg, 0.84 mmol) in a 2:1:1 mixture of THF/water/methanol (6mL) was added lithium hydroxide monohydrate (70 mg, 1.67 mmol) and thereaction stirred at room temperature for 2 h. Treated with 1 N HCl (1.6mL), partitioned between EtOAc and water (30 mL each), organic layerextracted with EtOAc (30 mL), dried (MgSO₄) and concentrated to give awhite foam (211 mg, 74% yield). This material was used in subsequentsteps without additional purification. ¹H NMR (400 MHz, DMSO-d₆) δ ppm12.43 (br. s., 1H) 7.19-7.41 (m, 1H) 4.05-4.28 (m, 1H) 3.91-4.05 (m, 2H)3.40-3.71 (m, 6H) 3.20-3.28 (m, 1H) 1.81-1.98 (m, 2H) 1.70-1.80 (m, 1H)1.46-1.69 (m, 2H) 1.32-1.46 (m, 1H) 0.65-1.03 (m, 10H).

Intermediate 104: methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-2-azaspiro[4.5]dec-3-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate

To a solution of2-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylicacid (103) (100 mg, 0.29 mmol), HATU (111 mg, 0.29 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (1) (168 mg, 0.29 mmol), prepared as described inExample 1 in anhydrous CH₂Cl₂ (2 mL), was added Hunig's base (0.2 mL,1.17 mmol) and the solution stirred at rt under nitrogen. After 1 h, thereaction was concentrated in vacuo, purified by C₁₈ RP chromatography,eluting with 10-90% ACN/water/0.2% NH₄OH, to afford the product as ayellow solid (133 mg, 55% yield).

Example 16 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8,8-dioxido-8-thia-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

A solution of methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8,8-dioxido-8-thia-2-azaspiro[4.5]dec-3-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate(108) (107 mg, 0.12 mmol) and ammonium acetate (94 mg, 1.2 mmol) indioxane (1.5 mL) was degassed with nitrogen and heated to 110° C. in asealed tube for 18 h. The reaction was cooled to room temperature anddiluted with ethyl acetate and filtered and concentrated in vacuo.Purified by SFC to afford the product as a tan solid (39 mg, 37% yield).¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.64 (br. s., 10H) 7.32 (br. s., 2H)5.05-5.24 (m, 1H) 4.04-4.29 (m, 1H) 3.99 (br. s., 1H) 3.81 (br. s., 1H)3.55-3.73 (m, 6H) 3.03-3.25 (m, 4H) 1.80-2.47 (m, 14H) 0.68-1.06 (m,16H). HRMS for C₄₄H₅₇N₈O₈S (M+H)⁺ calc: 857.4020. found: 857.4020.Purity (LC-MS): 97%.

Preparation of Intermediate 108

Intermediate 105: ethyl 8-thia-2-azaspiro[4.5]decane-3-carboxylate8,8-dioxide

This compound was prepared in 90% yield fromtetrahydro-2H-thiopyran-4-carbaldehyde 1,1-dioxide (1.05 g, 6.47 mmol)in an analogous fashion to Example 15.

Intermediate 106: ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-8-thia-2-azaspiro[4.5]decane-3-carboxylate8,8-dioxide

Intermediate 106 was prepared in 60% yield from ethyl8-thia-2-azaspiro[4.5]decane-3-carboxylate 8,8-dioxide (105) (200 mg,0.77 mmol) in an analogous fashion to Example 15.

Intermediate 107:2-{N-[(methyloxy)carbonyl]-L-valyl}-8-thia-2-azaspiro[4.5]decane-3-carboxylicacid 8,8-dioxide

Intermediate 107 was prepared in quantitative yield from ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-8-thia-2-azaspiro[4.5]decane-3-carboxylate8,8-dioxide (106) (187 mg, 0.45 mmol) in an analogous fashion to Example15. Used in subsequent steps without additional purification. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 12.48 (br. s., 1H) 7.13-7.44 (m, 1H) 4.10-4.31(m, 1H) 3.46-3.61 (m, 3H) 2.93-3.24 (m, 3H) 2.32 (dd, J=3.7, 1.76 Hz,1H) 1.93-2.13 (m, 3H) 1.73-1.94 (m, 2H) 1.55-1.71 (m, 1H) 0.66-1.04 (m,10H).

Intermediate 108: methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8,8-dioxido-8-thia-2-azaspiro[4.5]dec-3-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate

Prepared in 49% yield from2-{N-[(methyloxy)carbonyl]-L-valyl}-8-thia-2-azaspiro[4.5]decane-3-carboxylicacid 8,8-dioxide (107) (100 mg, 0.26 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl]-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (1) (148 mg, 0.26 mmol), in an analogous fashion asdescribed in Example 15.

Example 17 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[8,8-difluoro-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-azaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

A solution of ammonium acetate (540 mg, 6.9 mmol) and2-{4′-[({[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]carbonyl}oxy)acetyl]-4-biphenylyl}-2-oxoethyl(3S)-8,8-difluoro-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-azaspiro[4.5]decane-3-carboxylate(Intermediate 117) (307 mg, 0.35 mmol) in anhydrous dioxane (3.5 mL) wasdegassed with nitrogen and heated to 110° C. in a sealed tube for 3 h.The reaction was cooled to room temperature and partitioned betweenEtOAc and sat. NaHCO₃ (35 mL each), the organic layer was washed withbrine and dried (MgSO₄) and concentrated in vacuo. The residue waspurified by C₁₈ reverse phase chromatography eluting with 10-100%acetonitrile/water/0.2% N H₄OH to afford the title compound as a yellowsolid (235 mg, 80% yield). ¹H NMR (400 MHz, METHANOL-d₄) ppm 7.69-7.82(m, 3H) 7.63 (br. s., 5H) 7.17-7.37 (m, 2H) 5.03-5.19 (m, 1H) 4.20 (br.s., 1H) 4.06-4.16 (m, 1H) 3.96 (br. s., 1H) 3.85 (br. s., 1H) 3.62 (d,J=3.1 Hz, 6H) 2.24-2.38 (m, 3H) 2.15 (br. s., 3H) 1.85-2.09 (m, 8H)1.77-1.85 (m, 2H) 1.53-1.76 (m, 3H) 0.96 (br. s., 4H) 0.87 (d, J=6.3 Hz,12H). HRMS for C₄₅H₅₇N₈O₆F₂ (M+H)⁺ calc: 843.4369. found: 843.4371.Purity (LC-MS): 97%.

Preparation of Intermediate 70

Intermediate 60

(2S)-2-{[((2S)-2-{[(1,1-dimethylethyl)oxy]carbonyl]-1-pyrrolidinyl)carbonyl]amino}-3-methylbutanoicacid (24.57 g, 143 mmol) and N-[(methyloxy)carbonyl]-L-valine (25.1 g,143 mmol) were dissolved in DCM (50 mL). DIPEA (75 mL, 430 mmol) andHOBT (21.97 g, 143 mmol) were added. After 5 min EDC (27.5 g, 143 mmol)was added. The reaction was stirred at rt for 3 h. Diluted with water(50 mL) and 1N HCl (1 mL) was added. The precipitate was filtered offand organic/aqueous layers were filtered through a hydrophobic frit andconcentrated to dryness to afford 42.92 g of a colorless oil.

Intermediate 61

1,1-dimethylethyl N-[(methyloxy)carbonyl]-L-valyl-L-prolinate (61 g, 186mmol) was dissolved in HCl (50 ml, 1646 mmol) (150 mL0 of a 4M sol) andstirred for 5 h. Concentrated to dryness to afford 47.9 g of product asa light yellow sticky foam.

Intermediate 70

1,1′-(4,4′-biphenyldiyl)bis(2-bromoethanone) (37.8 g, 95 mmol) wasdissolved in DMF (800 mL) and degassed for 15 min (N2). Intermediate 61(21.99 g, 81 mmol) was dissolved in DMF (100 mL), followed by carefuladdition of NaH (2.94 g, 73.4 mmol, 60% in oil) under nitrogen over 15min. The solution was stirred under N2 for 15 min, then slowly addedover 15 min drop-wise to a solution of1,1′-(4,4′-biphenyldiyl)bis(2-bromoethanone), followed by stirring for 1h at RT. Solvent volume was then reduced in vacuo to ˜100 mL and cooledto 20° C. 100 mL of water was slowly added and resulting slightgrey-yellow solid was filtered and washed with water (200 mL), hexane(200 mL) and dried under the vacuum (12 h). The crude compound waspurified on 500 g of silica with hexane/ethyl acetate (increasinggradient from 50% to 100% EA), yielding 14.5 g (37.3%) of Intermediate70. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.11 (dd, J=12.0, 8.5 Hz, 4H) 7.96(d, J=8.4 Hz, 4H) 7.41 (d, J=8.4 Hz, 1H) 5.43-5.75 (m, 2H) 4.99 (s, 2H)4.53 (dd, J=8.6, 4.7 Hz, 1H) 4.03 (t, J=8.6 Hz, 1H) 3.76-3.90 (m, 1H)3.60-3.73 (m, 1H) 3.53 (s, 3H) 2.22-2.37 (m, 1H) 2.12-2.21 (m, 1H)1.85-2.06 (m, 3H) 0.90 (dd, J=10.7, 6.6 Hz, 6H).

Preparation of Intermediate 117

Intermediate 110: ethyl8-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-azaspiro[4.5]decane-3-carboxylate

This compound was obtained in 98% yield as a racemate from4-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}cyclohexanecarbaldehyde(mixture of cis/trans isomers) (7.35 g, 30.3 mmol) in an analogousfashion to Example 15.

Intermediate 111: 3-ethyl 2-(phenylmethyl)8-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-azaspiro[4.5]decane-2,3-dicarboxylate

To a solution of ethyl8-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-azaspiro[4.5]decane-3-carboxylate(110) (10.61 g, 31.3 mmol) dissolved in dry DCM, triethylamine was added(10.8 mL, 78 mmol), cooled to 0° C. followed by the addition of Cbz-Cl(6.2 mL, 43.5 mmol), and the reaction stirred at 0° C., for 5 min, rtfor 1 h. Reaction diluted with DCM (700 mL), washed with 0.1 N HCl (700mL), dried (MgSO₄) and concentrated in vacuo. The residue was purifiedby silica gel chromatography eluting with 10-60% hexanes/EtOAc to affordthe title compound as a yellow oil (5.73 g, 39% yield).

Intermediate 112: 3-ethyl 2-(phenylmethyl)8-hydroxy-2-azaspiro[4.5]decane-2,3-dicarboxylate

To a solution of 3-ethyl 2-(phenylmethyl)8-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-2-azaspiro[4.5]decane-2,3-dicarboxylate(111) (5.73 g, 12.05 mmol) in THF (60 mL) was added glacial acetic acid(1.38 mL, 24.0 mmol) followed by TBAF (24 mL) as a 1 M solution in THF.The reaction was stirred at room temperature for 72 h. The reaction waspartitioned between EtOAc and water (250 mL) each, the organic layer wasseparated and washed with saturated NaHCO₃ (100 mL) and dried (MgSO₄)and concentrated in vacuo. The reaction was found to be incomplete byTLC. The residue was dissolved in dry THF (60 mL), cooled to 0° C. andtreated with HF-pyridine (1.6 mL, 18.0 mmol), warmed to room temperatureand stirred for 2 h under nitrogen. The reaction was poured intosaturated NaHCO₃ (100 mL) and solid potassium carbonate was added untilgas evolution ceased. Extracted with EtOAc (2×150 mL), the organiclayers were combined and washed with 0.1 N HCl (100 mL), dried (MgSO₄)and concentrated in vacuo to afford the title compound in quantitativeyield as a yellow oil which was used in subsequent reactions withoutadditional purification.

Intermediate 113: 3-ethyl 2-(phenylmethyl)8-oxo-2-azaspiro[4.5]decane-2,3-dicarboxylate

To a solution of 3-ethyl 2-(phenylmethyl)8-hydroxy-2-azaspiro[4.5]decane-2,3-dicarboxylate (112) (4.36 g, 120.05mmol) in dry DCM (60 mL) was added Dess-Martin Periodinane (10.22 g,24.1 mmol) and the reaction stirred at rt under nitrogen for 18 h. Thereaction was poured into 10% aq sodium thiosulfate (150 mL) and sat.NaHCO₃ (150 mL) and stirred for 10 min. Extracted with DCM (2×150 mL),dried (MgSO₄) and concentrated in vacuo. The residue was purified bysilica gel chromatography eluting with 25-80% hexanes/EtOAc to affordthe title compound as a pale yellow oil (2.89 g, 67% yield).

Intermediate 114: 3-ethyl 2-(phenylmethyl)(3S)-8,8-difluoro-2-azaspiro[4.5]decane-2,3-dicarboxylate

To a solution of 3-ethyl 2-(phenylmethyl)8-oxo-2-azaspiro[4.5]decane-2,3-dicarboxylate (113) (2.89 g, 7.13 mmol)in anhydrous CH₂Cl₂ (50 mL) was added Deoxo-Fluor (2.2 mL, 12.1 mmol)followed by a catalytic amount of ethanol and the reaction stirred at rtunder nitrogen. After 2.5 h the reaction is poured into sat NaHCO₃ (150mL), stirred for 10 min. Extracted with DCM (2×150 mL), and the organiclayer was washed with 0.1 N HCl (100 mL), dried (MgSO₄) and concentratedin vacuo to afford the desired compound as a yellow oil (3.01 g) whichwas found to be contaminated with 3-ethyl 2-(phenylmethyl)(3S)-8-fluoro-2-azaspiro[4.5]dec-7-ene-2,3-dicarboxylate in a 1:1 ratio.The residue was dissolved in dry DCM (35 mL), and treated with mCPBA(77%, 1.66 g, 7.45 mmol) and stirred under nitrogen for 18 h. Thereaction was poured into saturated NaHCO₃ (40 mL) and 10% aqueous sodiumthiosulfate (40 mL) and stirred for 10 min. Extracted with DCM (100 mL)and dried (MgSO₄) and concentrated in vacuo. The residue was purified bysilica gel chromatography eluting with 5-50% hexanes/EtOAc. The racematewas then separated by chiral HPLC on a 10 μm OD column eluting with 25%isopropanol in hexanes to afford the title compound as a clear oil (632mg, 23% yield). The absolute configurations were determined byvibrational circular dichroism (VCD).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.41 (m, 5H) 4.98-5.23 (m, 2H)4.37 (ddd, J=19.3, 8.1, 8.0 Hz, 1H) 4.22 (q, J=7.2 Hz, 1H) 3.93-4.12 (m,1H) 3.45-3.70 (m, 1H) 3.36 (dd, J=10.8, 2.0 Hz, 1H) 2.22 (dd, J=12.8,8.5 Hz, 1H) 1.79-2.03 (m, 4H) 1.59-1.78 (m, 4H) 1.56 (br. s., 1H) 1.28(t, J=7.1 Hz, 1H) 1.22 (d, J=6.1 Hz, 1H) 1.13 (t, J=7.1 Hz, 1H). LC-MSESI (M+H)⁺=381.68.

Intermediate 115: ethyl(3S)-8,8-difluoro-2-azaspiro[4.5]decane-3-carboxylate

To a solution of 3-ethyl 2-(phenylmethyl)(3S)-8,8-difluoro-2-azaspiro[4.5]decane-2,3-dicarboxylate (114) (630 mg,1.65 mmol) in absolute ethanol (12 mL), was added 20% Pd(OH)₂ on carbon(65 mg) and the reaction hydrogenated on a Fisher-Porter apparatus for18 h at 60 psi. The reaction was filtered through celite andconcentrated in vacuo to afford the title compound as a clear oil (380mg, 93% yield).

Intermediate 116:(3S)-8,8-difluoro-2-{N-[(methyloxy)carbonyl]-L-valyl}-2-azaspiro[4.5]decane-3-carboxylicacid

To a solution of ethyl(3S)-8,8-difluoro-2-azaspiro[4.5]decane-3-carboxylate (115), (380 mg,1.54 mmol) in anhydrous CH₂Cl₂ (10 mL) was added HATU (614 mg, 1.6mmol), N-[(methyloxy)carbonyl]-L-valine (283 mg, 1.6 mmol) followed bytriethylamine (0.43 mL, 3.1 mmol) and the reaction stirred at roomtemperature under nitrogen for 1 h. The reaction was concentrated invacuo and the residue purified by silica gel chromatography eluting with15-80% hexanes/EtOAc. Appropriate fractions were combined andconcentrated in vacuo. The residue was dissolved in THF/water/methanol(5 mL/2.5 mL/2.5 mL) and lithium hydroxide monohydrate was added (119mg, 2.8 mmol) and the solution stirred at room temperature for 30 min.The reaction was treated with 1N HCl (3.5 mL) and partitioned betweenEtOAc and water (50 mL each). The aqueous layer was extracted with ethylacetate (50 mL), the organic layers were combined and dried (MgSO₄) andconcentrated in vacuo. The residue was triturated in diethyl ether andconcentrated in vacuo to afford the title compound as a white solid (542mg, 93% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.47 (br. s., 1H) 7.41(d, J=8.0 Hz, 1H) 4.24 (t, J=8.6 Hz, 1H) 3.95-4.14 (m, 2H) 3.52 (s, 3H)3.25-3.33 (m, 1H) 2.22 (dd, J=12.3, 8.4 Hz, 1H) 1.79-2.11 (m, 5H)1.58-1.77 (m, 3H) 1.41-1.59 (m, 2H) 0.93 (dd, J=12.7, 6.6 Hz, 6H). LC-MSESI (M+H)⁺=377.23.

Intermediate 117:2-{4′-[({[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]carbonyl}oxy)acetyl]-4-biphenylyl}-2-oxoethyl(3S)-8,8-difluoro-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-azaspiro[4.5]decane-3-carboxylate

To a solution of(3S)-8,8-difluoro-2-{N-[(methyloxy)carbonyl]-L-valyl}-2-azaspiro[4.5]decane-3-carboxylicacid (116) (168 mg, 0.45 mmol) and2-[4′-(bromoacetyl)-4-biphenylyl]-2-oxoethylN-[(methyloxy)carbonyl]-L-valyl-L-prolinate (70) (250 mg, 0.43 mmol) inanhydrous acetonitrile (2 mL) was added triethylamine (0.09 mL, 0.64mmol) and the reaction stirred at room temperature under nitrogen for 1h. The reaction was partitioned between EtOAc and 0.1 N HCl (30 mLeach), the organic layer was washed with brine and dried (MgSO₄) andconcentrated in vacuo. The residue was purified by silica gelchromatography eluting with 65-100% hexanes/EtOAc to afford the titlecompound as an off white solid (309 mg, 82% yield).

Example 18 dimethyl(4,4′-biphenyldiylbis{1H-imidazole-4,2-diyl(3S)-8-oxa-2-azaspiro[4.5]decane-3,2-diyl[(2S)-3-methyl-1-oxo-1,2-butanediyl]})biscarbamate

To a solution of bis(phenylmethyl)(3S,3'S)-3,3′-[4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl)]bis(8-oxa-2-azaspiro[4.5]decane-2-carboxylate)(119) (210 mg, 0.25 mmol) in trifluoroacetic acid (2 mL) cooled to 0° C.was added trifluoromethanesulfonic acid (0.13 mL) and the reactionwarmed to room temperature and stirred for 30 min. The reaction wasconcentrated in vacuo and rotovaped. The residue was rotovaped fromtoluene and the residue suspended in dichloromethane (3 mL) and treatedwith 4N HCl in dioxane (0.65 mL). The reaction was concentrated in vacuoand triturated in ether and filtered to obtain a brown solid.

To a solution of the solid in anhydrous DMF (2.5 mL) was addedN-[(methyloxy)carbonyl]-L-valine (88 mg, 0.5 mmol), HATU (183 mg, 0.48mmol) and triethylamine (0.4 mL, 2.88 mmol) and the reaction stirred atroom temperature for 1 h. The crude reaction mixture was purified byHPLC eluting with 10-90% acetonitrile/water/0.2% NH₄OH to afford thetitle compound as a pale yellow solid (79 mg, 37% yield). ¹H NMR (400MHz, METHANOL-d₄) δ ppm 7.60-7.79 (m, 10H) 7.33 (s, 2H) 5.11 (dd, J=9.9,7.9 Hz, 2H) 4.30 (d, J=10.2 Hz, 2H) 4.17 (d, J=8.2 Hz, 2H) 3.70-3.87 (m,6H) 3.56-3.71 (m, 10H) 2.40 (dd, J=12.9, 7.8 Hz, 2H) 2.13 (dd, J=12.6,10.3 Hz, 2H) 1.88-2.00 (m, 2H) 1.70-1.84 (m, 4H) 1.46-1.69 (m, 6H)0.83-0.97 (m, 12H). HRMS for C₄₈H₆₃N₈O₈ (M+H)⁺ calc: 879.4769. found:879.4769. Purity (LC-MS): 98%.

Preparation of Intermediate 119

Intermediate 118:3,3′-[4,4′-biphenyldiylbis(2-oxo-2,1-ethanediyl)]2,2′-bis(phenylmethyl)(3S,3'S)bis(-8-oxa-2-azaspiro[4.5]decane-2,3-dicarboxylate)

To a solution of 1,1′-(4,4′-biphenyldiyl)bis(2-bromoethanone) (155 mg,0.39 mmol) and(3S)-2-{[(phenylmethyl)oxy]carbonyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylicacid (121) (275 mg, 0.86 mmol) in anhydrous acetonitrile (4 mL) wasadded triethylamine (0.19 mL, 1.4 mmol) and the solution stirred at roomtemperature under nitrogen for 3.5 h. The reaction was partitionedbetween EtOAc and 0.1 N HCl (40 mL each), the organic layer washed withbrine and dried (MgSO₄) and concentrated in vacuo. The residue waspurified by silica gel chromatography eluting with 20-100% hexanes/EtOActo afford the title compound as a white solid (225 mg, 66% yield).

Intermediate 119: bis(phenylmethyl)(3S,3′S)-3,3′-[4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl)]bis(8-oxa-2-azaspiro[4.5]decane-2-carboxylate)

To a solution of3,3′-[4,4′-biphenyldiylbis(2-oxo-2,1-ethanediyl)]2,2′-bis(phenylmethyl)(3S,3'S)bis(-8-oxa-2-azaspiro[4.5]decane-2,3-dicarboxylate) (118) (225mg, 0.26 mmol) in dioxane (3.5 mL) was added ammonium acetate (318 mg,4.1 mmol). The reaction was degassed with nitrogen and heated to 110° C.in a sealed tube for 18 h. The reaction was partitioned between EtOAcand sat. NaHCO₃ (35 mL each), the organic layer was washed with brine,dried (MgSO₄) and concentrated in vacuo to afford the title compound inquantitative yield.

Preparation of Intermediate 121

Intermediate 120: 3-ethyl 2-(phenylmethyl)(3S)-8-oxa-2-azaspiro[4.5]decane-2,3-dicarboxylate

Ethyl 8-oxa-2-azaspiro[4.5]decane-3-carboxylate (3.54 g, 16.6 mmol) wasdissolved in dry dichloromethane (80 mL), triethylamine (5.8 mL, 41.5mmol) was added followed by benzylchloroformate (3.5 mL, 24.9 mmol) andthe reaction is stirred at room temperature for 2 h under nitrogen. Thereaction was diluted with dichloromethane, washed with 0.2 N HCl, driedover MgSO₄ and concentrated in vacuo. The residue was purified by silicagel chromatography eluting with 20-100% hexanes/EtOAc to afford a yellowoil. The racemate was then separated by chiral HPLC on a 10 μm OD columneluting with 25% isopropanol in hexanes to afford the title compound asa clear oil (1.66 g, 29% yield).

Intermediate 121:(3S)-2-{[(phenylmethyl)oxy]carbonyl}-8-oxa-2-azaspiro[4.5]decane-3-carboxylicacid

To a solution of 3-ethyl 2-(phenylmethyl)(3S)-8-oxa-2-azaspiro[4.5]decane-2,3-dicarboxylate (120) (300 mg, 0.86mmol) in THF/water/methanol (3 mL/1.5 mL/1.5 mL) was added lithiumhydroxide monohydrate (72 mg, 1.7 mmol) and the reaction stirred at roomtemperature for 30 min. The reaction was treated with 1N HCl (2 mL) andpartitioned between EtOAc and water. The aqueous layer was extractedwith EtOAc and the organic layer dried over MgSO₄ and concentrated invacuo to afford the title compound in quantitative yield. ¹H NMR (400MHz, DMSO-d₆) δ ppm 12.54-12.93 (m, 1H) 7.21-7.47 (m, 5H) 4.90-5.20 (m,2H) 4.11-4.41 (m, 1H) 3.44-3.70 (m, 5H) 3.24 (dd, J=15.0, 11.7 Hz, 1H)2.32 (td, J=13.6, 8.5 Hz, 1H) 1.73 (ddd, J=16.8, 12.9, 7.2 Hz, 1H)1.38-1.64 (m, 4H). LC-MS ESI (M−H)⁻=318.19.

Example 19 1,1-dimethylethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate

A solution of 1,1-dimethylethyl2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-3-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl]-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-2,8-diazaspiro[4.5]decane-8-carboxylate(134) (126 mg, 0.14 mmol) and ammonium acetate (105 mg, 1.4 mmol) inanhydrous dioxane (1.5 mL) was degassed with nitrogen and heated in asealed tube to 110° C. for 18 h. The reaction was concentrated in vacuoand purified by HPLC eluting with 10-90% acetonitrile/water/0.2% N H₄OHto afford the title compound as a tan solid (84 mg, 68% yield). ¹H NMR(400 MHz, METHANOL-d₄) δ ppm 7.56-7.87 (m, 10H) 7.31 (br. s., 2H)5.04-5.23 (m, 1H) 4.11-4.32 (m, 2H) 3.93-4.04 (m, 1H) 3.80-3.92 (m, 1H)3.63 (s, 6H) 3.33-3.61 (m, 6H) 1.86-2.49 (m, 9H) 1.68 (br. s., 1H) 1.56(br. s., 2H) 1.37-1.49 (m, 9H) 0.81-1.08 (m, 15H). HRMS for C₄₉H₆₅N₉O₈(M+H)⁺ calc: 908.5034. found: 908.5031. Purity (LC-MS): 93%.

Preparation of Intermediate 134

Intermediate 131: 8-(1,1-dimethylethyl) 3-ethyl2,8-diazaspiro[4.5]decane-3,8-dicarboxylate

This compound was obtained as a racemate from 1,1-dimethylethyl4-formyl-1-piperidinecarboxylate following the procedure outlined in WO98/08850 pp. 50.

Intermediate 132: 8-(1,1-dimethylethyl) 3-ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-2,8-diazaspiro[4.5]decane-3,8-dicarboxylate

To a solution of 8-(1,1-dimethylethyl) 3-ethyl2,8-diazaspiro[4.5]decane-3,8-dicarboxylate (131) (150 mg, 0.48 mmol),HATU (183 mg, 0.48 mmol) and N-[(methyloxy)carbonyl]-L-valine (93 mg,0.53 mmol) in anhydrous CH₂Cl₂ (4 mL) was added Hunig's base (0.17 mL,0.96 mmol) and the reaction stirred at room temperature under nitrogenfor 2 h. The reaction was concentrated in vacuo and purified by silicagel chromatography eluting with 15-80% hexanes/EtOAc to afford the titlecompound as a pale yellow oil (125 mg, 55% yield).

Intermediate 133:8-{[(1,1-dimethylethyl)oxy]carbonyl}-2-{N-[(methyloxy)carbonyl]-L-valyl}-2,8-diazaspiro[4.5]decane-3-carboxylicacid

To a solution of 8-(1,1-dimethylethyl) 3-ethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-2,8-diazaspiro[4.5]decane-3,8-dicarboxylate(132) (125 mg, 0.27 mmol) in THF/water/MeOH (1.2 mL/0.6 mL/0.6 mL) wasadded lithium hydroxide monohydrate (22 mg, 0.53 mmol) and the reactionstirred at room temperature for 1.5 h. The reaction was treated with 1NHCl (0.5 mL) and partitioned between EtOAc and 0.1 N HCl (10 mL each)and the aqueous layer extracted with EtOAc (10 mL). The organic layerswere combined and dried (MgSO₄) and concentrated in vacuo to afford thetitle compound as a white solid (107 mg, 91% yield). ¹H NMR (400 MHz,DMSO-d₆) δ ppm 12.29-12.54 (m, 1H) 7.08-7.44 (m, 1H) 4.47 (t, J=5.4 Hz,1H) 4.13-4.30 (m, 1H) 3.86-4.13 (m, 3H) 3.45-3.58 (m, 3H) 3.16-3.27 (m,2H) 2.64 (br. s., 1H) 2.15-2.31 (m, 1H) 1.81-1.95 (m, 1H) 1.43-1.67 (m,3H) 1.29-1.43 (m, 11H) 0.69-1.03 (m, 6H). LC-MS ESI (M−H)⁻=440.59.

Intermediate 134: 1,1-dimethylethyl2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-3-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl]-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-2,8-diazaspiro[4.5]decane-8-carboxylate

To a solution of8-{[(1,1-dimethylethyl)oxy]carbonyl}-2-{N-[(methyloxy)carbonyl]-L-valyl}-2,8-diazaspiro[4.5]decane-3-carboxylicacid (133) (106 mg, 0.24 mmol), HATU (91 mg, 0.24 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenyly]-1H-imidazol-2-ylyl-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride(1) (138 mg, 0.24 mmol) in anhydrous CH₂Cl₂ (4 mL) wasadded Hunig's base (0.17 mL, 0.96 mmol) and the reaction stirred at roomtemperature under nitrogen for 1 h. The reaction was concentrated invacuo and purified by HPLC eluting with 10-90% acetonitrile/water/0.2% NH₄OH to afford the title compound as an off-white solid (129 mg, 58%yield).

Example 20 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,8-diazaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

To a solution of 1,1-dimethylethyl2-{N-[(methyloxy)carbonyl]-L-valyl}-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate(68 mg, 0.08 mmol) in anhydrous CH₂Cl₂ (1 mL) was added trifluoroaceticacid (0.3 mL) and the reaction stirred at room temperature for 2 h. Thereaction was concentrated in vacuo and purified by HPLC eluting with10-90% acetonitrile/water/0.2% NH₄OH to afford the title compound as atan solid (49 mg, 81% yield). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm7.56-7.83 (m, 10H) 7.31 (br. s., 2H) 5.00-5.22 (m, 2H) 4.12-4.30 (m, 3H)4.01 (br. s., 1H) 3.80-3.91 (m, 1H) 3.57-3.70 (m, 6H) 2.76-3.07 (m, 4H)1.89-2.48 (m, 10H) 1.69-1.81 (m, 1H) 1.61 (br. s., 2H) 0.82-1.09 (m,15H). HRMS for C₄₄H₅₈N₉O₆ (M+H)⁺ calc: 808.4510. found: 808.4509. Purity(LC-MS): 94%.

Example 21 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[8-acetyl-2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,8-diazaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

To a solution of methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,8-diazaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(Example 20) (19 mg, 0.02 mmol) in anhydrous CH₂Cl₂ (0.5 mL) was addedtriethylamine (0.016 mL, 0.12 mmol) followed by acetyl chloride (0.01mL, 0.14 mmol) and the reaction stirred at room temperature for 1 h. Thereaction is concentrated in vacuo and dissolved in methanol (0.7 mL) towhich was added potassium carbonate (30 mg, 0.22 mmol) and the reactionwas stirred at room temperature for 2 h. The reaction was concentratedin vacuo and partitioned between CH₂Cl₂ and water (3 mL each) and theaqueous layer extracted with CH₂Cl₂ (3 mL) and the organic layerscombined and dried (MgSO₄) and concentrated in vacuo to afford the titlecompound as a yellow solid (18 mg, 90% yield). ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 7.54-7.88 (m, 10H) 7.32 (br. s., 2H) 5.05-5.23 (m,2H) 4.03-4.34 (m, 2H) 3.71-4.03 (m, 3H) 3.56-3.71 (m, 8H) 3.34-3.56 (m,2H) 2.14-2.46 (m, 4H) 1.89-2.14 (m, 6H) 1.46-1.83 (m, 4H) 1.19-1.32 (m,2H) 0.78-1.07 (m, 14H). HRMS for C₄₆H₆₀N₉O₇ (M+H)⁺ calc: 850.4616.found: 850.4617. Purity (LC-MS): 94%.

Example 22 methyl2-{N-[(methyloxy)carbonyl]-L-valyl}-3-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate

To a solution of methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,8-diazaspiro[4.5]dec-3-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(Example 20) (19 mg, 0.02 mmol) in anhydrous CH₂Cl₂ (0.5 mL) was addedtriethylamine (0.016 mL, 0.12 mmol) followed by acetyl chloride (0.011mL, 0.14 mmol) and the reaction stirred at room temperature for 1 h. Thereaction is concentrated in vacuo and dissolved in methanol (0.7 mL) towhich was added potassium carbonate (30 mg, 0.22 mmol) and the reactionwas stirred at room temperature for 2 h. The reaction was concentratedin vacuo and partitioned between CH₂Cl₂ and water (3 mL each) and theaqueous layer extracted with CH₂Cl₂ (3 mL) and the organic layerscombined and dried (MgSO₄) and concentrated in vacuo to afford the titlecompound as a yellow solid (15 mg, 74% yield). ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 7.51-7.92 (m, 10H) 7.32 (br. s., 2H) 5.02-5.25 (m,1H) 4.03-4.31 (m, 2H) 3.75-4.05 (m, 2H) 3.61-3.72 (m, 7H) 3.34-3.60 (m,4H) 1.82-2.50 (m, 7H) 1.70 (br. s., 2H) 1.44-1.63 (m, 4H) 1.27 (br. s.,4H) 0.77-1.10 (m, 14 H). HRMS for C₄₆H₆₀N₉O₈ (M+H)⁺ calc: 866.4565.found: 850.4564. Purity (LC-MS): 96%.

Example 23 1,1-dimethylethyl6-{N-[(methyloxy)carbonyl]-L-valyl}-7-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate

To a solution of 1,1-dimethylethyl6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-7-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate(148) (400 mg, 0.45 mmol) and ammonium acetate (343 mg, 4.5 mmol) inanhydrous dioxane (5 mL) was degassed with nitrogen and heated in asealed tube to 110° C. for 18 h. The reaction was diluted with EtOAc andfiltered and concentrated in vacuo. The residue was purified by C₁₈reverse phase chromatography eluting with 10-100%acetonitrile/water/0.2% NH₄OH to afford the title compound as a paleyellow solid (315 mg, 80% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm11.63-12.35 (m, 1H) 7.47-7.92 (m, 9H) 7.19-7.42 (m, 1H) 5.08 (br. s.,2H) 3.94-4.23 (m, 4H) 3.62-3.93 (m, 7H) 3.56 (s, 6H) 2.24-2.45 (m, 2H)2.14 (br. s., 2H) 1.77-2.07 (m, 6H) 1.37 (d, J=5.5 Hz, 9H) 0.76-1.02 (m,12H). HRMS for C₄₇H₆₂N₆O₈ (M+H)⁺ calc: 880.4721. found: 880.4725. Purity(LC-MS): 95%.

Preparation of Intermediate 148

Intermediate 145: 2-(1,1-dimethylethyl) 7-ethyl2,6-diazaspiro[3.4]octane-2,7-dicarboxylate

This compound was prepared from 1,1-dimethylethyl3-formyl-1-azetidinecarboxylate (650 mg, 3.5 mmol) in 93% yield in ananalogous fashion to Intermediate 131 in Example 19.

Intermediate 146: 2-(1,1-dimethylethyl) 7-ethyl6-{N-[(methyloxy)carbonyl]-L-valyl}-2,6-diazaspiro[3.4]octane-2,7-dicarboxylate

To a solution of 2-(1,1-dimethylethyl) 7-ethyl2,6-diazaspiro[3.4]octane-2,7-dicarboxylate (145) (500 mg, 1.76 mmol),HATU (735 mg, 1.93 mmol) and N-[(methyloxy)carbonyl]-L-valine (339 mg,1.93 mmol) in anhydrous CH₂Cl₂ (10 mL) was added Hunig's base (0.68 mL,3.87 mmol) and the reaction stirred at room temperature under nitrogenfor 2 h. The reaction was concentrated in vacuo and purified by C₁₈reverse-phase chromatography eluting with 10-90% ACN/water/0.2% NH₄OH toafford the title compound as an off-white solid (398 mg, 51% yield).

Intermediate 147:2-{[(1,1-dimethylethyl)oxy]carbonyl}-6-{N-[(methyloxy)carbonyl]-L-valyl}-2,6-diazaspiro[3.4]octane-7-carboxylicacid

To a solution of 2-(1,1-dimethylethyl) 7-ethyl6-{N-[(methyloxy)carbonyl]-L-valyl}-2,6-diazaspiro[3.4]octane-2,7-dicarboxylate(146) (344 mg, 0.78 mmol) in THF/water/methanol (3 mL/1.5 mL/1.5 mL) wasadded lithium hydroxide monohydrate (65 mg, 1.56 mmol) and the reactionstirred at room temperature for 1.5 h whereupon it was treated with 1NHCl (1.5 mL). The reaction was partitioned between EtOAc and water (30mL each), the aqueous layer was extracted with EtOAc (30 mL), theorganic layers were combined and dried (MgSO₄) and concentrated in vacuoto afford the title compound as a white solid (245 mg, 76% yield). ¹HNMR (400 MHz, DMSO-d₆) δ ppm 12.46 (br. s., 1H) 7.06-7.66 (m, 1H)4.13-4.32 (m, 1H) 3.88-4.12 (m, 2H) 3.56-3.89 (m, 5H) 3.39-3.57 (m, 3H)2.24-2.45 (m, 1H) 1.99-2.19 (m, 1H) 1.80-1.95 (m, 1H) 1.26-1.44 (m, 9H)0.56-1.00 (m, 6H). LC-MS ESI (M−H)⁻=412.41.

Intermediate 148: 1,1-dimethylethyl6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-7-({[2-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-2-oxoethyl]amino}carbonyl)-2,6-diazaspiro[3.4]octane-2-carboxylate

To a solution of2-{[(1,1-dimethylethyl)oxy]carbonyl}-6-{N-[(methyloxy)carbonyl]-L-valyl}-2,6-diazaspiro[3.4]octane-7-carboxylicacid (147) (245 mg, 0.59 mmol), HATU (225 mg, 0.59 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride(1) (342 mg, 0.59 mmol) in anhydrous CH₂Cl₂ (6 mL) wasadded Hunig's base (0.41 mL, 2.37 mmol) and the reaction stirred at roomtemperature under nitrogen for 1 h. The reaction was concentrated invacuo and purified by C₁₈ reverse phase chromatography eluting with10-100% acetonitrile/water/0.2% NH₄OH to afford the title compound as anoff-white solid (400 mg, 75% yield).

Example 24 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

This compound was prepared in an analogous fashion to Example 20 from1,1-dimethylethyl6-{N-[(methyloxy)carbonyl]-L-valyl}-7-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate(298 mg, 0.34 mmol) to afford the title compound as a yellow solid (203mg, 77% yield). ¹H NMR (400 MHz, METHANOL-d₄) 6 ppm 7.43-7.86 (m, 8H)7.04-7.42 (m, 2H) 5.20-5.41 (m, 1H) 5.01-5.19 (m, 2H) 4.30-4.43 (m, 1H)4.12-4.27 (m, 3H) 3.78-4.03 (m, 5H) 3.65-3.78 (m, 4H) 3.61-3.64 (m, 6H)3.39-3.52 (m, 1H) 2.54-2.77 (m, 1H) 2.38-2.54 (m, 1H) 2.09-2.39 (m, 2H)1.89-2.13 (m, 4H) 0.81-1.13 (m, 12H). HRMS for C₄₂H₅₄N₉O₆ (M+H)⁺ calc:780.4197. found: 780.4200. Purity (LC-MS): 96%.

Example 25 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[2-acetyl-6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

This compound was prepared in an analogous fashion to Example 21 frommethyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamateExample 24 (40 mg, 0.05 mmol) to afford the title compound as a yellowsolid (35 mg, 83% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.67-12.33(m, 1H) 7.19-7.99 (m, 12H) 5.07 (br. s., 1H) 3.92-4.25 (m, 4H) 3.67-3.94(m, 4H) 3.45-3.66 (m, 8H) 2.20-2.46 (m, 3H) 2.17 (br. s., 2H) 1.80-2.06(m, 5H) 1.60-1.82 (m, 3H) 0.78-1.02 (m, 12H). HRMS for C₄₄H₅₆N₉O₇ (M+H)⁺calc: 822.4303. found: 822.4300. Purity (LC-MS): 91%.

Example 26 methyl6-{N-[(methyloxy)carbonyl]-L-valyl}-7-(4-{4′-[2-((2S)-1-{N-[(methyloxy)carbonyl]-L-valyl}-2-pyrrolidinyl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate

This compound was prepared in an analogous fashion to Example 22 frommethyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(Example 24) (40 mg, 0.05 mmol) to afford the title compound as a yellowsolid (37 mg, 87% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.38-12.37(m, 1H) 7.58-7.93 (m, 8H) 7.19-7.59 (m, 3H) 4.93-5.17 (m, 1H) 3.63-4.22(m, 7H) 3.45-3.64 (m, 6H) 2.24-2.48 (m, 4H) 2.08 (br. s., 4H) 1.79-2.07(m, 6H) 0.99-1.18 (m, 1H) 0.75-0.97 (m, 14H). HRMS for C₄₄H₅₆N₉O₈ (M+H)⁺calc: 838.4252. found: 838.4252. Purity (LC-MS): 87%.

Example 27 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[2-[(methylamino)carbonyl]-6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

To a solution of methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl]-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(Example 24) (45 mg, 0.06 mmol) in anhydrous CH₂Cl₂ (0.6 mL) was addedmethyl isocyanate (0.01 mL, 0.17 mmol) and the reaction stirred at roomtemperature for 2 h. The solvent was removed in vacuo and the residuedissolved in methanol (1 mL) and potassium carbonate (40 mg, 0.29 mmol)was added and the reaction stirred at room temperature for 18 h. Thereaction was partitioned between CH₂Cl₂ (10 mL) and water (5 mL), theaqueous layer was extracted with CH₂Cl₂ (5 mL), the organic layers werecombined and dried (MgSO₄) and concentrated in vacuo to afford the titlecompound as a yellow solid (34 mg, 70% yield). ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 7.64 (br. s., 10H) 7.33 (br. s., 2H) 5.06-5.22 (m,1H) 4.03-4.46 (m, 3H) 3.73-4.04 (m, 8H) 3.54-3.73 (m, 6H) 3.46 (q, J=7.0Hz, 2H) 2.52-2.76 (m, 5H) 2.10-2.53 (m, 4H) 2.02 (br. s., 3H) 0.70-1.09(m, 12H). HRMS for C₄₄H₅₇N₁₀O₇ (M+H)⁺ calc: 837.4412. found: 838.4416.Purity (LC-MS): 91%.

Example 28 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-(methylsulfonyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

To a solution of methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2,6-diazaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate(Example 24) (60 mg, 0.08 mmol) in anhydrous CH₂Cl₂ (1 mL) was addedtriethylamine (0.054 mL, 0.39 mmol) and the reaction was cooled to 0° C.Methanesulfonyl chloride (0.018 mL, 0.23 mmol) was added and thereaction stirred at 0° C. for 15 minutes. The solvent was removed invacuo and the residue dissolved in methanol (1 mL) and potassiumcarbonate (80 mg, 0.58 mmol) was added and the reaction stirred at roomtemperature for 1.5 h. The reaction was partitioned between CH₂Cl₂ (10mL) and water (10 mL) the organic layer was dried (MgSO₄) andconcentrated in vacuo to afford the title compound as a tan solid (57mg, 86% yield). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50-7.86 (m, 10H)7.33 (br. s., 2H) 5.06-5.21 (m, 1H) 4.04-4.26 (m, 2H) 3.75-4.05 (m, 6H)3.56-3.72 (m, 6H) 3.38-3.55 (m, 2H) 2.84-3.04 (m, 3H) 2.70-2.86 (m, 1H)2.40-2.64 (m, 1H) 2.10-2.40 (m, 3H) 1.83-2.09 (m, 2H) 1.20-1.37 (m, 2H)1.07-1.20 (m, 2H) 0.81-1.07 (m, 12H). HRMS for C₄₃H₆₆N₉O₈S (M+H)⁺ calc:858.3973. found: 858.3975. Purity (LC-MS): 86%.

Example 29 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[(7S)-2,2-difluoro-6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6-azaspiro[3.4]oct-7-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

To a solution of2-{4′-[({[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]carbonyl}oxy)acetyl]-4-biphenylyl}-2-oxoethyl(7S)-2,2-difluoro-6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6-azaspiro[3.4]octane-7-carboxylate(157) (102 mg, 0.12 mmol) in anhydrous dioxane (1.2 mL) was addedammonium acetate (184 mg, 2.4 mmol) and the reaction heated to 110° C.for 4 h. The reaction was partitioned between EtOAc and saturatedNaHCO₃, the organic layer was washed with brine and dried over MgSO₄ andconcentrated in vacuo. The residue was purified by C₁₈ reverse phasechromatography eluting with 10-100% acetonitrile/water/0.2% N H₄OH toafford the title compound as a yellow solid (68 mg, 69% yield). ¹H NMR(400 MHz, DMSO-d₆) δ ppm 11.42-12.37 (m, 2H) 7.56-7.90 (m, 8H) 7.51 (s,2H) 7.19-7.46 (m, 2H) 4.73-5.39 (m, 2H) 3.91-4.23 (m, 3H) 3.82 (br. s.,2H) 3.54 (s, 6H) 2.55-2.86 (m, 4H) 2.20-2.45 (m, 2H) 2.14 (br. s., 2H)1.73-2.09 (m, 5H) 0.57-0.99 (m, 12H). HRMS for C₄₃H₅₃N₈O₆F₂ (M+H)⁺ calc:815.4056. found: 815.4059. Purity (LC-MS): 97%.

Preparation of Intermediate 157

Intermediate 150: ethyl2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-6-azaspiro[3.4]octane-7-carboxylate

This compound was prepared in an analogous fashion to 110 (example 17)from 3-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}cyclobutanecarbaldehyde(2.23 g, 10.4 mmol) to afford the title compound (2.97 g, 92% yield) asa yellow oil.

Intermediate 151: 7-ethyl 6-(phenylmethyl)2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-6-azaspiro[3.4]octane-6,7-dicarboxylate

This compound was prepared in an analogous fashion to 111 (Example 17)from ethyl2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-6-azaspiro[3.4]octane-7-carboxylate(150) (2.97 g, 9.5 mmol) to obtain the title compound as a yellow oil(1.87 g, 44% yield).

Intermediate 152: 7-ethyl 6-(phenylmethyl)2-hydroxy-6-azaspiro[3.4]octane-6,7-dicarboxylate

To a solution of 7-ethyl 6-(phenylmethyl)2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}-6-azaspiro[3.4]octane-6,7-dicarboxylate(151) (1.87 g, 4.2 mmol) in THF (20 mL) was added glacial acetic acid(0.48 mL), followed by TBAF (8.5 mL, 1M solution in THF) and thereaction heated to 45° C. for 18 h. The reaction was concentrated invacuo and partitioned between EtOAc and water. The organic layer waswashed with saturated NaHCO₃ followed by brine, and then dried overMgSO₄ and concentrated in vacuo to afford the title compound as a clearoil in quantitative yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.17-7.48 (m,5H) 4.83-5.15 (m, 3H) 3.88-4.34 (m, 4H) 3.35-3.47 (m, 2H) 2.02-2.39 (m,3H) 1.65-1.96 (m, 3H) 1.00-1.22 (m, 3H). LC-MS ESI (M+H)⁺=334.17.

Intermediate 153: 7-ethyl 6-(phenylmethyl)2-oxo-6-azaspiro[3.4]octane-6,7-dicarboxylate

This compound was prepared in an analogous fashion to 113 (Example 17)from 7-ethyl 6-(phenylmethyl)2-hydroxy-6-azaspiro[3.4]octane-6,7-dicarboxylate (152) (1.39 g, 4.2mmol) to give the title compound (1.25 g, 90% yield) as a clear oil.

Intermediate 154: 7-ethyl 6-(phenylmethyl)(7S)-2,2-difluoro-6-azaspiro[3.4]octane-6,7-dicarboxylate

To a solution of 7-ethyl 6-(phenylmethyl)2-oxo-6-azaspiro[3.4]octane-6,7-dicarboxylate (153) (1.25 g, 3.8 mmol)in anhydrous dichloromethane (20 mL) was added Deoxo-Fluor (1.2 mL, 6.4mmol) followed by ethanol (0.04 mL, 0.75 mmol) and the reaction stirredat room temperature under nitrogen for 18 h. The reaction is poured intosaturated NaHCO₃ and stirred for 10 min. The mixture is extracted withdichloromethane (2×) and the organic layer was washed with 0.1 N HCl anddried over MgSO₄ and concentrated in vacuo. The residue was purified bysilica gel chromatography eluting with 5-50% hexanes/EtOAc. The racematewas then separated by chiral HPLC on a 10 μm OD column eluting with 25%isopropanol in hexanes to afford the title compound as a clear oil (356mg, 30% yield).

Intermediate 155: ethyl(7S)-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxylate

This compound was prepared in an analogous fashion to 115 (Example 17)from 7-ethyl 6-(phenylmethyl)(7S)-2,2-difluoro-6-azaspiro[3.4]octane-6,7-dicarboxylate (14) (356 mg,1.0 mmol) to afford the title compound as a clear oil (209 mg, 95%yield).

Intermediate 156:(7S)-2,2-difluoro-6-{N-[(methyloxy)carbonyl]-L-valyl}-6-azaspiro[3.4]octane-7-carboxylicacid

This compound was prepared in an analogous fashion to 116 (Example 17)from ethyl (7S)-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxylate (155)(207 mg, 0.94 mmol) to afford the title compound as a white solid (263mg, 81% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.53 (br. s., 1H) 7.46(d, J=8.0 Hz, 1H) 4.24 (t, J=7.8 Hz, 1H) 4.02 (d, J=10.4 Hz, 1H) 3.92(t, J=8.5 Hz, 1H) 3.62 (d, J=10.4 Hz, 1H) 3.51 (s, 3H) 2.54-2.78 (m, 4H)2.28-2.44 (m, 1H) 1.81-2.05 (m, 2H) 0.91 (dd, J=11.2, 6.7 Hz, 6H). LC-MSESI (M+H)⁺=349.13.

Intermediate 157:2-{4-[({[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]carbonyl}oxy)acetyl]-4-biphenylyl}-2-oxoethyl(7S)-2,2-difluoro-6-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-6-azaspiro[3.4]octane-7-carboxylate

This compound was prepared in an analogous fashion to 117 (Example 17)from(7S)-2,2-difluoro-6-{N-[(methyloxy)carbonyl]-L-valyl}-6-azaspiro[3.4]octane-7-carboxylicacid (156) (59 mg, 0.17 mmol) and2-[4′-(bromoacetyl)-4-biphenylyl]-2-oxoethylN-[(methyloxy)carbonyl]-L-valyl-L-prolinate (70) (95 mg, 0.16 mmol) toafford the title compound as a white solid (104 mg, 75% yield).

Example 30 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-1-azaspiro[4.5]dec-2-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

To a solution of methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-1-azaspiro[4.5]dec-2-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate(169) (71 mg, 0.09 mmol) in anhydrous dioxane (1 mL) was added ammoniumacetate (66 mg, 0.86 mmol) and the reaction was degassed with nitrogenand heated to 110° C. for 48 h in a sealed tube. The reaction waspurified by HPLC eluting with 10-90% water/acetonitrile/0.2% NH₄OH toafford the title compound as an off-white solid (10 mg, 14% yield). ¹HNMR (400 MHz, METHANOL-d₄) δ ppm 7.56-7.86 (m, 9H) 7.16-7.44 (m, 2H)6.88-7.09 (m, 1H) 5.72 (br. s., 1H) 5.09-5.21 (m, 1H) 4.03-4.28 (m, 1H)3.80-4.04 (m, 6H) 3.42-3.71 (m, 9H) 2.11-2.56 (m, 8H) 1.88-2.11 (m, 6H)0.80-1.06 (m, 12H). HRMS for C₄₄H₅₇N₈O₇ (M+H)⁺ calc: 809.4350. found:809.4347. Purity (LC-MS): 93%.

Preparation of Intermediate 169

Intermediate 162: ethyl2-[(diphenylmethylidene)amino]-4-(tetrahydro-4H-pyran-4-ylidene)butanoate

To a solution of ethyl N-(diphenylmethylidene)glycinate (5.29 g, 19.8mmol), benzyltriethylammonium chloride (0.41 g, 1.8 mmol) and cesiumhydroxide monohydrate (4.54 g, 27.0 mmol) in anhydrous dichloromethane(50 mL) was added 4-(2-bromoethylidene)tetrahydro-2H-pyran (3.44 g, 18.0mmol) as a solution in anhydrous dichloromethane (40 mL) and thereaction stirred at room temperature for 72 h under nitrogen. Thereaction was partitioned between dichloromethane and water and theaqueous layer was extracted with dichloromethane. The organic layer wasdried over sodium sulfate and concentrated in vacuo. The residue waspurified by silica gel chromatography eluting with 5-40% hexanes/EtOActo afford the title compound as a clear oil in quantitative yield.

Intermediate 163: ethyl2-amino-4-(tetrahydro-4H-pyran-4-ylidene)butanoate

To a solution of ethyl2-[(diphenylmethylidene)amino]-4-(tetrahydro-4H-pyran-4-ylidene)butanoate(162) (6.8 g, 18.0 mmol) in THF (30 mL) was added water (30 mL) andglacial acetic acid (20 mL) and the reaction stirred at room temperaturefor 2.5 h. The reaction was concentrated in vacuo and the residuedissolved in 0.1 N HCl. It was extracted twice with ethyl acetate andthe organic layer was discarded. To the aqueous layer was added solidpotassium carbonate until the solution gave blue pH paper. The aqueouslayer was extracted with 15% isopropanol/dichloromethane (3×) and theorganic layer dried over sodium sulfate and concentrated in vacuo toafford the title compound as a clear oil (3.05 g, 79% yield).

Intermediate 164: ethyl2-{[(4-nitrophenyl)sulfonyl]amino}-4-(tetrahydro-4H-pyran-4-ylidene)butanoate

To a solution of ethyl2-amino-4-(tetrahydro-4H-pyran-4-ylidene)butanoate (163) (1.0 g, 4.7mmol) in anhydrous dichloromethane (30 mL) was added Hunig's base (1.6mL, 9.4 mmol) followed by 4-nitrobenzenesulfonyl chloride (1.14 g, 5.2mmol) and the reaction stirred at room temperature under nitrogen for 2h. The reaction was diluted with dichloromethane and washed with 0.1 NHCl, the organic layer was dried over MgSO₄ and concentrated in vacuo.The residue was purified by silica gel chromatography eluting with10-70% hexanes/EtOAc to afford the title compound as an off-white solidin quantitative yield.

Intermediate 165: ethyl1-[(4-nitrophenyl)sulfonyl]-8-oxa-1-azaspiro[4.5]decane-2-carboxylate

To a solution of ethyl2-{[(4-nitrophenyl)sulfonyl]amino}-4-(tetrahydro-4H-pyran-4-ylidene)butanoate(164) (1.87 g, 4.7 mmol) in anhydrous chloroform (47 mL) was addedtrifluoromethanesulfonic acid (0.2 mL, 2.3 mmol) and the reactionstirred at room temperature under nitrogen for 4 h. The reaction wasdiluted with dichloromethane and washed with saturated NaHCO₃ and theorganic layer dried over MgSO₄ and concentrated in vacuo. The residuewas purified by silica gel chromatography eluting with 10-70%hexanes/EtOAc to afford the title compound as a white solid (1.47 g, 79%yield). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.19-8.36 (m, 2H) 8.08 (d,J=8.8 Hz, 2H) 4.52 (dd, J=8.5, 2.1 Hz, 1H) 3.99-4.14 (m, 2H) 3.87-3.99(m, 2H) 3.21-3.49 (m, 2H) 2.58-2.78 (m, 2H) 2.10-2.30 (m, 2H) 1.89-2.06(m, 2H) 1.77 (dd, J=13.0, 2.1 Hz, 1H) 1.35 (dd, J=12.9, 1.6 Hz, 1H) 1.22(t, J=7.1 Hz, 3H). LC-MS ESI (M+H)⁺=399.47.

Intermediate 166: ethyl 8-oxa-1-azaspiro[4.5]decane-2-carboxylate

To a solution of ethyl1-[(4-nitrophenyl)sulfonyl]-8-oxa-1-azaspiro[4.5]decane-2-carboxylate(165) (1.47 g, 3.7 mmol) in anhydrous acetonitrile (25 mL) was addedpotassium carbonate (0.76 g, 5.5 mmol), 18-crown-6 (0.2 g, 0.7 mmol) andthiophenol (0.6 mL. 5.5 mmol) and the reaction stirred at roomtemperature for 18 h. The reaction was concentrated in vacuo and theresidue dissolved in 0.1 N HCl. The aqueous layer was extracted twicewith EtOAc and the organic layer was discarded. The aqueous layer wastreated with solid potassium carbonate until the solution gave blue pHpaper. It was extracted with 15% isopropanol/dichloromethane (3×) andthe organic layer dried over sodium sulfate and concentrated in vacuo toafford the title compound as a clear oil (0.71 g, 90% yield).

Intermediate 167: ethyl1-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-1-azaspiro[4.5]decane-2-carboxylate

To a solution of ethyl 8-oxa-1-azaspiro[4.5]decane-2-carboxylate (166)(125 mg, 0.59 mmol) in anhydrous dichloromethane (2 mL) was added silvercyanide (98 mg, 0.73 mmol) followed by N-[(methyloxy)carbonyl]-L-valylchloride (142 mg, 0.73 mmol) as a solution in anhydrous dichloromethane(3.5 mL) and the solution stirred at room temperature for 18 h undernitrogen. The reaction is filtered and treated with methanol and stirredfor 5 minutes. The reaction is concentrated in vacuo and the residuepurified by silica gel chromatography eluting with 30-100% hexanes/EtOActo afford the title compound as a white solid (43 mg, 20% yield).

Intermediate 168:1-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-1-azaspiro[4.5]decane-2-carboxylicacid

To a solution of ethyl1-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-1-azaspiro[4.5]decane-2-carboxylate(167) (80 mg, 0.2 mmol) in THF/water/methanol (1.0 mL/0.5 mL/0.5 mL) wasadded lithium hydroxide monohydrate (13 mg, 0.3 mmol) and the reactionstirred at room temperature for 72 h. The reaction was treated with 1NHCl (0.5 mL) and the reaction partitioned between EtOAc and 0.1 N HCl.The organic layer was separated and dried over MgSO₄ and concentrated invacuo to afford the title compound as a white solid (53 mg, 72% yield).¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.88 (br. s., 1H) 7.45-7.61 (m, 1H)4.89-5.03 (m, 1H) 3.70-3.87 (m, 2H) 3.65 (t, J=9.2 Hz, 1H) 3.53 (s, 3H)3.02 (td, J=12.9, 4.9 Hz, 1H) 2.71-2.90 (m, 1H) 2.22-2.38 (m, 1H)1.87-2.19 (m, 3H) 1.53-1.73 (m, 1H) 1.32-1.42 (m, 1H) 1.06-1.15 (m, 1H)0.61-0.98 (m, 8H). LC-MS ESI (M+H)⁺=342.95.

Intermediate 169: methyl((1S)-2-methyl-1-{[(2S)-2-(4-{4′-[({[1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8-oxa-1-azaspiro[4.5]dec-2-yl]carbonyl}amino)acetyl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}propyl)carbamate

To a solution of1-{N-[(methyloxy)carbonyl]-L-valyl}-8-oxa-1-azaspiro[4.5]decane-2-carboxylicacid (168) (52 mg, 1.5 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl]-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (1) (88 mg, 0.15 mmol) and HATU (58 mg, 0.15 mmol) inanhydrous dichloromethane (1.2 mL) was added Hunig's base (0.12 mL, 0.68mmol) and the reaction stirred at room temperature for 1 h. The reactionwas concentrated in vacuo and purified by HPLC eluting with 10-90%water/acetonitrile/0.2% NH₄OH to afford the title compound as anoff-white solid (73 mg, 58% yield).

Example 31 methyl((1S)-1-{[(2S)-2-(4-{4′-[2-(1-acetyl-8-oxa-1-azaspiro[4.5]dec-2-yl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}-2-methylpropyl)carbamate

This compound was prepared in an analogous fashion to Example 30 frommethyl{(1S)-1-[((2S)-2-{4-[4′-({[(1-acetyl-8-oxa-1-azaspiro[4.5]dec-2-yl)carbonyl]amino}acetyl)-4-biphenylyl]-1H-imidazol-2-yl]-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamate(172) (116 mg, 0.16 mmol) to afford the title compound as a tan solid(19 mg, 17% yield). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.51-7.90 (m,8H) 7.17-7.52 (m, 2H) 5.08-5.26 (m, 2H) 3.81-4.03 (m, 4H) 3.64 (s, 3H)3.41-3.61 (m, 3H) 2.21-2.51 (m, 5H) 2.01-2.21 (m, 4H) 1.97 (d, J=19.9Hz, 5H) 1.77-1.91 (m, 1H) 1.64-1.75 (m, 1H) 1.31-1.42 (m, 1H) 0.79-1.04(m, 8H). HRMS for C₃₉H₄₈N₇O₅ (M+H)⁺ calc: 694.3717. found: 694.3718.Purity (LC-MS): 79%.

Preparation of Intermediate 172

Intermediate 170: ethyl1-acetyl-8-oxa-1-azaspiro[4.5]decane-2-carboxylate

To a solution of ethyl 8-oxa-1-azaspiro[4.5]decane-2-carboxylate (166)(135 mg, 0.63 mmol) in anhydrous dichloromethane (2 mL) was addedHunig's base (0.22 mL, 1.3 mmol) followed by acetyl chloride (0.05 mL,0.76 mmol) and the reaction stirred at room temperature under nitrogenfor 1.5 h. The reaction was diluted with dichloromethane and washed with0.1 N HCl and the organic layer dried over MgSO₄ and concentrated invacuo. The residue was purified by silica gel chromatography elutingwith 60-100% hexanes/EtOAc to afford the title compound as a yellow oil(94 mg, 58% yield).

Intermediate 171: 1-acetyl-8-oxa-1-azaspiro[4.5]decane-2-carboxylic acid

To a solution of ethyl1-acetyl-8-oxa-1-azaspiro[4.5]decane-2-carboxylate (170) (93 mg, 0.34mmol) in THF/water/methanol (1.1 mL/0.6 mL/0.6 mL) was added lithiumhydroxide monohydrate (29 mg, 0.69 mmol) and the reaction stirred atroom temperature for 3 h. The reaction is treated with 1N HCl (0.7 mL)and partitioned between EtOAc and 0.1 N HCl, the aqueous layer wasextracted twice with EtOAc, the combined organic layers were dried overMgSO₄ and concentrated in vacuo to afford the title compound as a whitesolid (79 mg, 95% yield).

Intermediate 172: methyl{(1S)-1-[((2S)-2-{4-[4′-({[(1-acetyl-8-oxa-1-azaspiro[4.5]dec-2-yl)carbonyl]amino}acetyl)-4-biphenylyl]-1H-imidazol-2-yl}-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamate

This compound was prepared in an analogous fashion to 169 from1-acetyl-8-oxa-1-azaspiro[4.5]decane-2-carboxylic acid (171) (56 mg,0.25 mmol) and methyl{(1S)-1-[((2S)-2-{4-[4′-(aminoacetyl)-4-biphenylyl]-1H-imidazol-2-yl]-1-pyrrolidinyl)carbonyl]-2-methylpropyl}carbamatedihydrochloride (1) (142 mg, 0.25 mmol) to afford the title compound asan off-white solid (118 mg, 67% yield).

Examples 32 to 37 were prepared, using the synthetic sequence similar toExamples 30 and 31.

Example 32 methyl[(1S)-1-({(2S)-2-[4-(4′-{2-[8,8-difluoro-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1-azaspiro[4.5]dec-2-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)-2-methylpropyl]carbamate

¹H NMR (400 MHz, METHANOL-d₄) δ 7.92-7.51 (m, 8H), 7.29 (br. s., 2H),5.71 (br. s., 1H), 5.32 (br. s., 1H), 4.21 (d, J=7.4 Hz, 1H), 4.15-3.80(m, 4H), 3.77-3.41 (m, 8H), 3.18 (br. s., 1H), 3.12-2.82 (m, 1H),2.63-1.40 (m, 16H), 1.05-0.84 (m, 10H), 0.78-0.64 (m, 1H), 0.44-0.28 (m,1H). HRMS for C45H57F2N8O6 (M+H)⁺ calc: 843.4369. found: 843.4368.

Example 33 methyl[(1S)-1-({8,8-difluoro-2-[4-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-azaspiro[4.5]dec-1-yl}carbonyl)propyl]carbamate

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.84 (d, J=7.8 Hz, 3H), 7.72-7.41 (m,6H), 7.31-7.09 (m, 3H), 5.73-5.17 (m, 4H), 5.12 (br. s., 1H), 4.4]-4.06(m, 3H), 3.97-3.56 (m, 11H), 3.37-2.76 (m, 4H), 2.60-1.55 (m, 9H),1.46-1.20 (m, 2H), 1.06 (d, J=6.0 Hz, 2H), 1.00-0.79 (m, 5H), 0.56 (d,J=7.3 Hz, 1H). HRMS for C44H55F2N8O6 (M+H)⁺ calc: 829.4213. found:829.4211.

Example 34 methyl((1S)-2-{8,8-difluoro-2-[4-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-azaspiro[4.5]dec-1-yl}-1-methyl-2-oxoethyl)carbamate

¹H NMR (400 MHz, CHLOROFORM-d) δ 10.97-10.14 (m, 2H), 7.91-7.40 (m, 8H),7.34-7.16 (m, 2H), 5.68-5.15 (m, 3H), 5.08 (d, J=6.0 Hz, 1H), 4.34 (d,J=8.0 Hz, 2H), 3.95-3.54 (m, 8H), 3.37-2.69 (m, 4H), 2.60-1.57 (m, 13H),1.56-1.18 (m, 3H), 1.06 (d, J=6.8 Hz, 1H), 0.89 (d, J=6.5 Hz, 6H). HRMSfor C43H53F2N8O6 (M+H)⁺ calc: 815.4056. found: 815.4061.

Example 35 methyl[(1S)-1-({8,8-difluoro-2-[4-(4′-{2-[(2S)-1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-2-pyrrolidinyl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-azaspiro[4.5]dec-1-yl}carbonyl)-3-methylbutyl]carbamate

¹H NMR (400 MHz, CHLOROFORM-d) δ 11.07-9.94 (m, 2H), 8.17-7.42 (m, 10H),7.33-7.08 (m, 2H), 5.72-5.19 (m, 3H), 5.14-4.99 (m, 1H), 4.86 (br. s.,1H), 4.50-4.09 (m, 2H), 4.02-3.50 (m, 8H), 3.45 (s, 1H), 3.33-2.71 (m,4H), 2.59-1.47 (m, 11H), 1.40 (br. s., 3H), 1.19-0.67 (m, 11H),0.63-0.40 (m, 1H).). HRMS for C46H59F2N8O6 (M+H)⁺ calc: 857.4526. found:857.4531.

Example 36 methyl((1S)-1-{[(2S)-2-(4-{4′-[2-(1-acetyl-8,8-difluoro-1-azaspiro[4.5]dec-2-yl)-1H-imidazol-4-yl]-4-biphenylyl}-1H-imidazol-2-yl)-1-pyrrolidinyl]carbonyl}-2-methylpropyl)carbamate

¹H NMR (400 MHz, CHLOROFORM-d) δ 11.17-10.07 (m, 2H), 8.16-7.40 (m,10H), 7.35-7.03 (m, 2H), 5.85-4.98 (m, 2H), 4.59-4.12 (m, 1H), 3.72 (s,5H), 3.46 (br. s., 1H), 3.33-2.72 (m, 2H), 2.76-1.39 (m, 17H), 1.27-0.74(m, 5H). HRMS for C40H48F2N704 (M+H)⁺ calc: 728.3736. found: 728.3736.

Example 37 methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[1-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-8,8-dioxido-8-thia-1-azaspiro[4.5]dec-2-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate

¹H NMR (400 MHz, CHLOROFORM-d) δ 7.92-7.52 (m, 9H), 5.39 (br. s., 4H),4.36 (br. s., 1H), 3.98-3.82 (m, 1H), 3.82-3.60 (m, 10H), 3.19 (br. s.,4H), 2.88-2.70 (m, 1H), 2.33 (br. s., 4H), 2.02 (s, 4H), 1.81 (br. s.,5H), 1.06 (s, 1H), 0.89 (d, J=7.0 Hz, 6H), 0.74 (d, J=6.5 Hz, 3H),0.44-0.34 (m, 3H). HRMS for C44H57N808S (M+H)⁺ calc: 857.4020. found:857.4018.

Protocol for Testing and Data Analysis of Compounds in the HCV RepliconAssay

Compounds were assayed for activity against HCV using the genotype 1aand 1b subgenomic replicon model systems. Stable cell lines bearing thegenotype 1a and 1b replicons were used for screening of compounds. Bothreplicons are bicistronic and contain the firefly luciferase gene. TheET cell line is stably transfected with RNA transcripts harboring aI₃₈₉luc-ubi-neo/NS3-3′/ET replicon with fireflyluciferase-ubiquitin-neomycin phosphotransferase fusion protein andEMCV-IRES driven NS3-5B polyprotein containing the cell culture adaptivemutations (E1202G; T12801; K1846T) (Krieger at al, 2001 andunpublished). The genotype 1a replicon is a stable cell line licensedfrom Apath LLC, modified to contain the firefly luciferase gene. Thecells were grown in DMEM, supplemented with 10% fetal calf serum, 2 mMGlutamine, Penicillin (100 IU/mL)/Streptomycin (100 μg/mL), 1×nonessential amino acids, and 250-500 μg/mL G418 (“Geneticin”). Theywere all available through Life Technologies (Bethesda, Md.). The cellswere plated at 5×10³ cells/well in 384 well plates containing compounds.The final concentration of compounds ranged between 0.03 pM to 50 μm andthe final DMSO concentration of 0.5-1%.

Luciferase activity was measured 48 hours later by adding a Steady gloreagent (Promega, Madison, Wis.). Percent inhibition of replication datawas plotted relative to no compound control. Under the same condition,cytotoxicity of the compounds was determined using cell titer glo(Promega, Madison, Wis.). EC50s were determined from an 11 point doseresponse curve using 3-4-fold serial dilution for each compound, whichspans a concentration range >1000 fold. The level of inhibition for eachcompound was determined with Activity Base or with BioAssay plus theExcel XC50 module. Percent inhibition was determined with the followingequation where the cross-talk corrected value is the value from the testwell, the compound positive control mean is the average value of thewells with no compound present, and the DMSO negative control mean isthe average value of the wells with DMSO but no cells present.

$\frac{100*\left( \begin{pmatrix}{{{Cross}\text{-}{talk}\mspace{14mu} {corrected}\mspace{14mu} {value}} -} \\{{Compound}\mspace{14mu} {Positive}\mspace{14mu} {Control}\mspace{14mu} {Mean}}\end{pmatrix} \right)}{\begin{matrix}{{{DMSO}\mspace{14mu} {Negative}\mspace{14mu} {Control}\mspace{14mu} {Mean}} -} \\{{Compound}\mspace{14mu} {Positive}\mspace{14mu} {Control}\mspace{14mu} {Mean}}\end{matrix}}$

These normalized values are exported to EC₅₀ where they are plottedagainst the molar compound concentrations using the standard fourparameter logistic equation:

$y = {A + \frac{B - A}{1 + \left\lbrack \frac{10^{X}}{10^{c}} \right\rbrack^{D}}}$

Where:

A=minimum y D=slope factorB=maximum y x=log₁₀ compound concentration [M]C=log₁₀EC₅₀ pEC₅₀=−C

As shown below, all tested compounds, except for Example 20, were foundto inhibit the activity of the replicon with pEC₅₀ >5.

Replicon Replicon 1A pEC₅₀ 1B pEC₅₀ Example 1 8.8 11.0 Example 2 10.411.1 Example 3 7.6 10.8 Example 4 10.4 11.0 Example 5 10.1 10.7 Example6 10.6 11.1 Example 7 9.1 11.6 Example 8 10.5 11.1 Example 9 10.5 11.2Example 10 8.7 11.0 Example 11 8.9 11.0 Example 12 10.1 11.4 Example 1310.2 11.4 Example 14 9.2 11.3 Example 15 10.2 10.8 Example 16 9.4 10.4Example 17 10.4 11.6 Example 18 10.7 10.9 Example 19 8.0 10.4 Example 20<7.5 8.8 Example 21 8.0 9.7 Example 22 9.1 10.8 Example 23 8.9 11.0Example 24 8.5 9.9 Example 25 8.9 9.8 Example 26 8.7 10.3 Example 27 8.29.6 Example 28 8.8 10.3 Example 29 10.0 11.5 Example 30 8.8 10.2 Example31 9.5 10.9 Example 32 8.8 10.8 Example 33 9.0 10.8 Example 34 9.2 11.0Example 35 8.7 10.9 Example 36 7.6 10.0 Example 37 7.9 9.5

Preparation of crystalline salts of the compound of Example 2: methyl[(1S)-2-methyl-1-({(2S)-2-[4-(4′-{2-[(8S)-7-((2S)-3-methyl-2-{[(methyloxy)carbonyl]amino}butanoyl)-1,4-dioxa-7-azaspiro[4.4]non-8-yl]-1H-imidazol-4-yl}-4-biphenylyl)-1H-imidazol-2-yl]-1-pyrrolidinyl}carbonyl)propyl]carbamate.

Crystalline Di-HCl Salt of the Compound of Example 2:

WO 2009/020828 discloses the crystalline di-HCl salt of a compound saidto be useful for treating HCV infection. The title compound of WO2009/020828 was prepared as described in that patent.

The compound of Example 2 was isolated and purified as the amorphousfree-base. This amorphous free-base was dissolved in acetone (3.6 mL; 30vol) at room temperature with stirring. Hydrochloric acid (316 uL of1.0M in dioxane; 2.1 equivalents) was added and resulted in an amorphousprecipitate. Methanol (480 uL; 4 vol) was added in aliquots until solidsjust dissolved. Crystalline seeds of the WO 2009/020828 di-HCl compoundwere added, and the mixture stirred over the weekend. The product wasfiltered with no wash, and the yield was 62.6% (84.0 mg; 0.0946 mmol) ofcrystalline di-HCl salt of the compound of Example 2.

Crystalline Di-HCl Salt of the Compound of Example 2:

The compound of Example 2 was isolated and purified as the amorphousfree-base. This amorphous free-base (555 mg; 0.696 mmol) was dissolvedin acetone (8.3 mL; 15 vol) and methanol (2.2 mL; 4 vol) at 50° C. withstirring. Hydrochloric acid (1.46 mL of 1.0M in dioxane; 2.1equivalents) was added slowly followed by seed crystals of the di-HClsalt of the compound of Example 2. The slurry was maintained at 50° C.for one hour, cooled to room temperature, and stirred over the weekend.The product was quickly filtered with no wash and dried at 50° C. in avacuum oven with nitrogen bleed. The yield was 54.0% (327 mg; 0.376mmol) of crystalline di-HCl salt of the compound of Example 2.

Crystalline Sulfate Salt of the Compound of Example 2:

The amorphous sulfate salt of the compound of Example 2 was prepared byadding 1.0 eq of sulfuric acid to a solution of free-base andconcentrating to dryness. The amorphous sulfate salt (˜50 mg; 0.056mmol) was taken up in acetone (750 ul; 15 volumes), and the mixture washeated to 50° C. Methanol (210 ul; 4.2 vol) was added 10 uL at a timeuntil the solids almost all dissolved, resulting in a cloudy solution.This cloudy solution was mixed at 50° C. for 16 hours, then cooled to23° C. The product was filtered, analyzed, and determined to beconsistent with crystalline sulfate salt of the compound of Example 2.

Crystalline Sulfate Salt of the Compound of Example 2:

The amorphous free-base of the compound of Example 2 (250 mg; 0.314mmol) was mixed with methanol (1.25 mL; 5 vol) and heated to 50° C. withstirring to accelerate dissolution. Sulfuric acid (0.105 mL of 3.0M inwater; 1.0 equivalent) was added slowly followed by seed crystals of thesulfate salt of the compound of Example 2. The slurry was maintained at50° C. for three hours to form a moderately thick, yellow slurry. Thetemperature was decreased to 15° C. to increase the yield. The productwas filtered with no wash and dried at 50° C. in a vacuum oven withnitrogen bleed. The yield was 73.6% (211 mg; 0.231 mmol) of crystallinesulfate salt of GSK2336805.

Analysis of Crystalline Salts:

The crystalline di-HCl and sulfate salts of the compound of Example 2were analyzed by ion chromatography, powder X-ray diffraction (PXRD),Raman, DSC, and TGA. All analyses were consistent with crystallinesalts.

The powder X-ray diffraction was performed with a PANalytical X′Pert-ProMPD with Johansson Kα1 monochromator, using X′Celerator detector. Thekey operating parameters were: Radiation: Cu (Kα1), 1.54060 angstroms(monochromatic); Detector: X′Celerator; Tension: 45 kV; Current: 40 mA;Start angle: 2.0° 20; End angle: 50.0° 20; Step size: 0.02°; Time/step:40.0 sec; Scan speed: 0.05°/sec; Incident beam: 2° fixed anti-scatterslit, and programmable divergence slit; Diffracted beam: 0.02 rad sollerslit, and programmable anti-scatter slit. Samples were prepared onsilicon zero background sample holder.

The di-HCl salt of the compound of Example 2 had significant peaks inpowder X-ray diffraction pattern at values of two theta in degrees andd-spacing in Angstrom in parenthesis of 5.3 (16.55), 9.9 (8.94), 10.4(8.54), 13.3 (6.65), 18.9 (4.70), 20.3 (4.37), 21.2 (4.18), 22.5 (3.95),23.2 (3.84), 23.7 (3.75), 24.4 (3.65), 26.3 (3.39), 27.6 (3.23). The TGAtrace of the di-HCl salt of the compound of Example 2 was consistentwith an anhydrous form. The DSC trace of the di-HCl salt of the compoundof Example 2 showed an onset of melting and/or decomposition atapproximately 262° C.

The sulfate salt of the compound of Example 2 had significant peaks inpowder X-ray diffraction pattern at values of two theta in degrees andd-spacing in Angstrom of 5.6 (15.87), 7.1 (12.39), 8.0 (11.10), 10.5(8.41), 11.9 (7.41), 12.6 (7.02), 13.4 (6.61), 14.3 (6.18), 16.6 (5.33),17.5 (5.07), 18.4 (4.81), 20.0 (4.43), 21.2 (4.19), 23.8 (3.73). The TGAtrace of the sulfate salt of the compound of Example 2 was consistentwith a variable hydrate form. The DSC trace of the sulfate salt of thecompound of Example 2 showed an onset of melting and/or decomposition atapproximately 241° C.

What is claimed is:
 1. A method for treating an HCV viral infection in ahuman comprising administration the compound

or a pharmaceutically acceptable salt thereof, and the administration ofat least one other antiviral agent.
 2. The method of claim 1 wherein thedepicted compound is enantiomerically enriched with the followingenantiomer


3. The method of claim 1 wherein said salt is either a di-HCl salt or asulfate salt.
 4. The method of claim 1 wherein said salt is a sulfatesalt.
 5. The method of claim 2 wherein said salt is either a di-HCl saltor a sulfate salt.
 6. The method of claim 2 wherein said salt is asulfate salt.